U.S. patent application number 09/954692 was filed with the patent office on 2003-02-06 for methods and compositions for polypeptide engineering.
This patent application is currently assigned to Maxygen, Inc.. Invention is credited to Patten, Phillip A., Stemmer, Willem P. C..
Application Number | 20030027156 09/954692 |
Document ID | / |
Family ID | 25084330 |
Filed Date | 2003-02-06 |
United States Patent
Application |
20030027156 |
Kind Code |
A1 |
Patten, Phillip A. ; et
al. |
February 6, 2003 |
Methods and compositions for polypeptide engineering
Abstract
Methods are provided for the evolution of proteins of industrial
and pharmaceutical interest, including methods for effecting
recombination and selection. Compositions produced by these methods
are also disclosed.
Inventors: |
Patten, Phillip A.; (Menlo
Park, CA) ; Stemmer, Willem P. C.; (Los Gatos,
CA) |
Correspondence
Address: |
QUINE INTELLECTUAL PROPERTY LAW GROUP, P.C.
P O BOX 458
ALAMEDA
CA
94501
US
|
Assignee: |
Maxygen, Inc.
Redwood City
CA
|
Family ID: |
25084330 |
Appl. No.: |
09/954692 |
Filed: |
September 12, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09954692 |
Sep 12, 2001 |
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08769062 |
Dec 18, 1996 |
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6335160 |
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08769062 |
Dec 18, 1996 |
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08621859 |
Mar 25, 1996 |
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6117679 |
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08769062 |
Dec 18, 1996 |
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08564955 |
Nov 30, 1995 |
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5811238 |
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Current U.S.
Class: |
435/6.14 ;
435/183; 435/455; 435/69.1 |
Current CPC
Class: |
C12N 9/16 20130101; C40B
40/02 20130101; G01N 33/6845 20130101; C12N 15/1058 20130101; C12N
9/0069 20130101; C12N 15/64 20130101; C12N 7/00 20130101; C12N
2795/14143 20130101; C12Q 1/6853 20130101; C12Q 1/686 20130101;
C07K 2317/565 20130101; C12N 9/86 20130101; C12N 15/1031 20130101;
C12N 15/1034 20130101; C12N 15/1037 20130101; C12N 2795/14043
20130101; C12Q 1/6811 20130101; C07K 16/00 20130101; C12N 15/1027
20130101; A61K 38/45 20130101; C12N 9/1007 20130101; C40B 40/08
20130101; C07K 14/43595 20130101; C07K 14/56 20130101; C07K
2317/622 20130101; G01N 33/68 20130101; C12N 15/67 20130101; C07K
14/545 20130101; C12N 15/10 20130101; A61K 48/00 20130101; C12N
15/52 20130101; C40B 50/06 20130101 |
Class at
Publication: |
435/6 ; 435/69.1;
435/183; 435/455 |
International
Class: |
C12Q 001/68; C12N
009/00; C12N 015/87 |
Claims
What is claimed is:
1. A method for evolving a protein encoded by a DNA substrate
molecule comprising: (a) digesting at least a first and second DNA
substrate molecule, wherein the at least a first and second
substrate molecules differ from each other in at least one
nucleotide, with a restriction endonuclease; (b) ligating the
mixture to generate a library of recombinant DNA molecules; (c)
screening or selecting the products of (b) for a desired property;
and (d) recovering a recombinant DNA substrate molecule encoding an
evolved protein.
2. The method of claim 1, wherein the restriction endonuclease
generates non-palindromic ends at cleavage sites.
3. The method of claim 1, wherein the substrate molecules have been
engineered to contain at least one recognition site for a
restriction endonuclease having non-palindromic ends at cleavage
sites.
4. The method of claim 1, wherein (a)-(d) are repeated.
5. The method of claim 1, wherein the DNA substrate molecule
comprises a gene cluster.
6. The method of claim 1, wherein at least one restriction
endonuclease fragment from a DNA substrate molecule is isolated and
subjected to mutagenesis to generate a library of mutant
fragments.
7. The method of step 6, wherein the library of mutant fragments is
used in the ligation of (b).
8. The method of claim 7, wherein the DNA substrate molecule
encodes all or part of a protein selected from Table I.
9. The method of claim 6, wherein mutagenesis comprises recursive
sequence recombination.
10. The method of claim 1, wherein the products of (d) are
subjected to mutagenesis.
11. The method of claim 10, wherein mutagenesis comprises recursive
sequence recombination.
12. The method of claim 1, wherein the products of (d) are used as
a DNA substrate molecule in (b).
13. The method of claim 10, wherein the products of claim 10 are
used in (d).
14. The method of claim 1, wherein the recombinant DNA substrate
molecule of (d) comprises a library of recombinant DNA substrate
molecules.
15. An evolved protein produced by the method of claim 1.
16. A method for evolving a protein encoded by a DNA substrate
molecule by recombining at least a first and second DNA substrate
molecule, wherein the at least a first and second substrate
molecules differ from each other in at least one nucleotide and
comprise defined segments, the method comprising: (a) providing a
set of oligonucleotide PCR primers, comprising at least one primer
for each strand of each segment, wherein the primer sequence is
complementary to at least one junction with another segment; (b)
amplifying the segments of the at least a first and second DNA
substrate molecules with the primers of step (a) in a polymerase
chain reaction; (c) assembling the products of step (b) to generate
a library of recombinant DNA substrate molecules; (d) screening or
selecting the products of (c) for a desired property; and (e)
recovering a recombinant DNA substrate molecule from (d) encoding
an evolved protein.
17. The method of claim 16, wherein the at least a first and second
DNA substrate molecules are subjected to mutagenesis prior to step
(a).
18. The method of claim 16, wherein the at least a first and second
DNA substrate molecules comprise alleles of a gene.
19. The method of claim 16, wherein the at least a first and second
DNA substrate molecules comprise a library of mutants.
20. The method of claim 16, wherein the segments are defined by
sites within intergenic regions.
21. The method of claim 16, wherein the segments are defined by
sites within introns.
22. The method of claim 16, wherein the primers comprise a uracil
substitution at one or more thymidine residues.
23. The method of claim 22, wherein the products of (b) are treated
with uracil glycosylase.
24. The method of claim 16, wherein (a)-(e) are repeated.
25. The method of claim 16, wherein the at least a first and second
DNA substrate molecule comprises a gene cluster.
26. The method of claim 16, wherein the at least first and second
DNA substrate molecule encodes all or part of a DNA polymerase.
27. The method or claim 16, wherein at least one PCR primer differs
from the at least a first and second DNA substrate molecules in at
least one nucleotide.
28. The method of claim 27, wherein the PCR primer comprises a
nucleotide sequence of a known mutant or polymorphism of the at
least a first or second DNA substrate molecule.
29. The method of claim 28, wherein the PCR primer is degenerate
and encodes the nucleotide sequences of more than one known mutant
or polymorphism of the at least a first or second DNA substrate
molecule.
30. The method of claim 29, wherein the at least a first and second
DNA substrate molecule encodes all or part of a protein selected
from Table I.
31. The method of claim 17, wherein mutagenesis comprises recursive
sequence recombination.
32. The method of claim 16, wherein the products of (e) are
subjected to mutagenesis.
33. The method of claim 32, wherein mutagenesis comprises recursive
sequence recombination.
34. The method of claim 32, wherein the products of claim 32 are
used in (b).
35. The method of claim 16, wherein the products of (e) are used as
a DNA substrate molecule in (b).
36. The method of claim 16, wherein the recombinant DNA substrate
molecule of e) comprises a library of recombinant DNA substrate
molecules.
37. An evolved protein produced by ate method of claim 16.
38. A method of enriching a population of DNA fragments for mutant
sequences comprising: (a) denaturing and renaturing the population
of fragments to generate a population of hybrid double-stranded
fragments in which at least one double-stranded fragment comprises
at least one base pair mismatch; (b) fragmenting the products of
(a) into fragments of about 20-100 bp; (c) affinity-purifying
fragments having a mismatch on an affinity matrix to generate a
pool of DNA fragments enriched for mutant sequences; and (d)
assembling the products of (c) to generate a library of recombinant
DNA substrate molecules.
39. The method of claim 38, wherein the population of DNA fragments
is derived from at least a first and second DNA substrate molecule,
the at least a first and second DNA substrate molecule differing
from each other in at least one nucleotide.
40. The method of claim 39, wherein the at least a first and second
DNA substrate molecules are obtained by mutagenesis of a DNA
substrate molecule.
41. The method of claim 39, wherein the at least a first and second
DNA substrate molecules comprise alleles of a gene.
42. The method of claim 39, wherein the at least a first and second
DNA substrate molecules comprise polymorphic variants of a
gene.
43. The method of claim 38, wherein the DNA substrate molecule
encodes all or part of a protein selected from Table I.
44. The method of claim 38, wherein the products of are mixed with
the products of (a) prior to (d).
45. A method for evolving a protein encoded by a DNA substrate
molecule, by recombining at least a first and second DNA substrate
molecule, wherein the at least a first and second substrate
molecules share a region of sequence homology of about 10 to 100
base pairs and comprise defined segments, the method comprising:
(a) providing regions of homology in the at least a first and
second DNA substrate molecules by inserting an intron sequence
between at least two defined segments; (b) fragmenting and
recombining DNA substrate molecules of (a), wherein regions of
homology are provided by the introns; (c) screening or selecting
the products of (b) for a desired property; and (d) recovering a
recombinant DNA substrate molecule from the products of (c)
encoding an evolved protein.
46. The method of claim 45, wherein the introns are
self-splicing.
47. The method of claim 45, wherein the inserted introns comprise
from about 1 to about 10 nonhomologous introns.
48. The method of claim 45, wherein the intron comprises a
recognition site for a restriction endonucleases having
non-palindromic ends at cleavage sites.
49. The method of claim 45, wherein (b)-(d) are repeated.
50. The method of claim 45, wherein the DNA substrate molecule
comprises a gene cluster.
51. The method of claim 45, wherein at least one segment from a DNA
substrate molecule is isolated and subjected to mutagenesis to
generate a library of mutant fragments.
52. The method of claim 45, wherein the library of mutant segments
is used in the recombination of (b).
53. The method of claim 45, wherein the segments are defined by
exons.
54. The method of claim 45, wherein the segments are defined by
intergenic regions.
55. The method of claim 45, wherein the at least a first and second
DNA substrate molecules encode protein homologues.
56. The method of claim 45, wherein the intron contains a lox site,
and wherein the products of (b) are used to transfect a Cre.sup.30
host.
57. The method of claim 45, wherein the at least a first and second
DNA substrate molecule encodes all or part of a protein selected
from Table I.
58. The method of claim 45, wherein the at least a first and second
DNA substrate molecule are subjected to mutagenesis prior to step
(a).
59. The method of claim 58, wherein mutagenesis comprises recursive
sequence recombination.
60. The method of claim 45, wherein the products of (d) are
subjected to mutagenesis.
61. The method of claim 58, wherein mutagenesis comprises recursive
sequence recombination.
62. The method of claim 45, wherein the products of (d) are used as
a DNA substrate molecule (b).
63. The method of claim 45, wherein the recombinant DNA substrate
molecule of (d) comprises a library of recombinant DNA substrate
molecules.
64. An evolved protein produced by the method of claim 45.
65. A method for evolving a protein encoded by a DNA substrate
molecule by recombining at least a first and second DNA substrate
molecule, wherein the at least a first and second substrate
molecules differ from each other in at least one nucleotide and
comprise defined segments, the method comprising: (a) providing a
set of oligonucleotide PCR primers, wherein for each junction of
segments a pair of primers is provided, one member of each pair
bridging the junction at one end of a segment and the other
bridging the junction at the other end of the segment, with the
terminal ends of the DNA molecule having as one member of the pair
a generic primer, and wherein a set of primers is provided for each
of the at least a first and second substrate molecules; (b)
amplifying the segments of the at least a first and second DNA
substrate molecules with the primers of (a) in a polymerase chain
reaction; (c) assembling the products of (b) to generate a pool of
recombinant DNA molecules; (d) selecting or screening the products
of (c) for a desired property; and (e) recovering a recombinant DNA
substrate molecule from the products of (d) encoding an evolved
protein.
66. The method of claim 65, wherein (a)-(e) is repeated.
67. The method of claim 65, wherein the at least a first and second
DNA substrate molecules are subjected to mutagenesis prior to
(a).
68. The method of claim 65, wherein the at least a first and second
DNA substrate molecule comprise sequences encoding protein
homologues.
69. The method of claim 65, wherein the primers comprise a uracil
substitution ac one or more thymidine residues.
70. The method of claim 69, wherein the products of (b) are treated
with uracil glycosylase.
71. The method of claim 65, wherein the at least a first and second
DNA substrate molecule encodes all or part of a protein selected
from Table I.
72. The method of claim 65, wherein the at least a first and second
DNA substrate molecule comprises a gene cluster.
73. An evolved protein produced by the method of claim 65.
74. The method of claim 65, wherein at least one PCR primer differs
from the at least a first and second substrate molecules in at
least one nucleotide.
75. The method of claim 74, wherein the PCR primer comprises a
nucleotide sequence of a known mutant or polymorphism of the at
least a first or second substrate molecule.
76. The method of claim 75, wherein the PCR primer is degenerate
and encodes the nucleotide sequences of more than one known mutant
or polymorphism of the at least a first or second substrate
molecule.
77. The method of claim 67, wherein mutagenesis comprises recursive
sequence recombination.
78. The method of claim 65, wherein the products of (e) are
subjected to mutagenesis.
79. The method of claim 78, wherein mutagenesis comprises recursive
sequence recombination.
80. The method of claim 65, wherein the products of (e) are used as
a DNA substrate molecule in (b).
81. The method of claim 65, wherein the recombinant DNA substrate
molecule of (e) comprises a library of recombinant DNA substrate
molecules.
82. A method for optimizing expression of a protein by evolving the
protein, wherein the protein is encoded by a DNA substrate
molecule, comprising: (a) providing a set of oligonucleotides,
wherein each oligonucleotide comprises at least two regions
complementary to the DNA molecule and at least one degenerate
region, each degenerate region encoding a region of an amino acid
sequence of the protein; (b) assembling the set of oligonucleotides
into a library of full length genes; (c) expressing the products of
(b) in a host cell; (d) screening the products of (c) for improved
expression of the protein; and (e) recovering a recombinant DNA
substrate molecule encoding an evolved protein from (d).
83. The method of claim 82, wherein the primers comprise about 20
nucleotides complementary to the DNA substrate molecule followed by
a second region of about 20 degenerate nucleotides of homology with
the DNA substrate molecules followed by about 20 nucleotides
complementary to the DNA substrate.
84. The method claim 82, wherein the protein is bovine intestinal
alkaline phosphatase.
85. The method of claim 84, wherein the oligonucleotides comprise
one or more primers from Table II.
86. The method of claim 82, wherein the DNA substrate molecule
encodes all or part of a protein selected from Table I.
87. The method of claim 82, wherein the DNA molecule comprises a
gene cluster.
88. The method of claim 82, wherein (a)-(e) are repeated.
89. The method of claim 82, wherein the oligonucleotides comprise
at least 5' and 3' nucleotide complementary to the DNA substrate
molecule and about 20-300 nucleotides having up to about 85%
sequence homology with a region of the DNA substrate molecule.
90. The method of claim 89, wherein the oligonucleotides comprise a
set of oligonucleotides in which each oligonucleotide overlaps with
a second oligonucleotide.
91. The method of claim 82, wherein the products of (e) are
subjected to mutagenesis.
92. The method of claim 91, wherein mutagenesis comprises recursive
sequence recombination.
93. The method of claim 82, wherein the recombinant DNA substrate
molecule of (e) comprises a library of recombinant DNA substrate
molecules.
94. An evolved protein produced by the method of claim 82.
95. A method for optimizing expression of a protein encoded by a
DNA substrate molecule by evolving the protein, wherein the DNA
substrate molecule compresses at least one lac operator and a
fusion of a DNA sequence encoding the protein with a DNA sequence
encoding a lac headpiece dimer, the method comprising: (a)
transforming a host cell with a library of mutagenized DNA
substrate molecules; (b) inducing expression of the protein encoded
by the library of (a); (c) preparing an extract of the product of
(b); (d) fractionating insoluble protein from complexes of soluble
protein and DNA; and (e) recovering a DNA substrate molecule
encoding an evolved protein from (d).
96. The method of claim 95, wherein (a)-(e) are repeated.
97. The method of claim 95, wherein the DNA substrate molecule
encodes all or part of a protein selected from Table I.
98. An evolved protein produced by the method of claim 95.
99. The method of claim 95, wherein the products of (e) are
subjected to mutagenesis.
100. The method of claim 99, wherein mutagenesis comprises
recursive sequence recombination.
101. The method of claim 95, wherein the products of (e) are used
as a DNA substrate molecule in (a).
102. The method of claim 95, wherein the recombinant DNA substrate
molecule of (e) comprises a library of recombinant DNA substrate
molecules.
103. A method for evolving functional expression of a protein
encoded by a DNA substrate molecule comprising a fusion of a DNA
sequence encoding the protein with a DNA sequence encoding
filamentous phage protein to generate a fusion protein, the method
comprising: (a) providing a host cell producing infectious
particles expressing a fusion protein encoded by a library of
mutagenized DNA substrate molecules; (b) recovering from (a)
infectious particles displaying the fusion protein; (c) affinity
purifying particles displaying the mutant protein using a ligand
for the protein; and (d) recovering a DNA substrate molecule
encoding an evolved protein from affinity purified particles of
(c).
104. The method of claim 103, wherein (a)-(d) are repeated.
105. The method of claim 103, wherein the DNA substrate molecule
encodes all or part of a protein selected from Table I.
106. An evolved protein produced by the method of claim 103.
107. The method of claim 103, wherein the products of (d) are
subjected to mutagenesis.
108. The method of claim 107, wherein mutagenesis comprises
recursive sequence recombination.
109. The method of claim 107, wherein the products of claim 107 are
used as a DNA substrate molecule in (a).
110. The method of claim 103, wherein the DNA substrate molecule of
(e) comprises a library of DNA substrate molecules.
111. The method of claim 103, wherein DNA sequence encoding the
filamentous phage protein comprises a phagemid.
112. The method of claim 103, wherein DNA sequence encoding the
filamentous phage protein comprises a phage.
113. A method for optimizing expression of a protein encoded by a
DNA substrate molecule comprising a fusion of a DNA sequence
encoding the protein with a DNA substrate encoding a lac headpiece
dimer, wherein the DNA substrate molecule is present on a first
plasmid vector, the method comprising: (a) providing a host cell
transformed with the first vector and a second vector comprising a
library of mutants of at least one chaperonin geneand at least one
lac operator; (b) preparing an extract of the product of (a); (c)
fractionating insoluble protein from complexes of soluble protein
and DNA; and (d) recovering DNA encoding a chaperonin gene from
(c).
114. The method of claim 113, wherein the DNA substrate molecule
encodes all or part of a protein selected from Table I.
115. The method of claim 113, wherein the DNA substrate is
subjected to mutagenesis independently of the chaperonin gene prior
to (a).
116. The method of claim 113, wherein the DNA of (d) comprises a
library of mutants.
117. The method of claim 113, wherein the first and second vectors
are the same vector.
118. The method of claim 113, wherein (d) further comprises
recovering an evolved DNA substrate molecule from the products of
(c).
119. An evolved chaperonin produced by the method of claim 113.
120. An evolved protein produced by the method of claim 113.
121. The method of claim 113, wherein (a)-(d) are repeated.
122. The method of claim 113, wherein the products of (d) are
subjected to mutagenesis.
123. The method of claim 122, wherein mutagenesis comprises
recursive sequence recombination.
124. The method of claim 122, wherein the products of claim 122 are
used in (a).
125. A method for optimizing expression of a protein encoded by a
DNA substrate molecule comprising a fusion of a DNA sequence
encoding the protein with a filamentous phage gene, wherein the
fusion is carried on a phagemid comprising a library of chaperonin
gene mutants, the method comprising: (a) providing a host cell
producing infectious particles expressing a fusion protein encoded
by a library of mutagenized DNA substrate molecules; (b) recovering
from (a) infectious particles displaying the fusion protein; (c)
affinity purifying particles displaying the protein using a ligand
for the protein; and (d) recovering DNA encoding the mutant
chaperonin from affinity purified particles of (c).
126. The method of claim 125, wherein (a)-(d) are repeated.
127. The method of claim 125, wherein the DNA substrate molecule
encodes all or part of a protein selected from Table I.
128. An evolved chaperonin produced by the method of claim 125.
129. An evolved protein produced by the method of claim 125.
130. The method of claim 125, wherein the products of (d) are
subjected to mutagenesis.
131. The method of claim 130, wherein mutagenesis comprises
recursive sequence recombination.
132. The method of claim 130, wherein the products of claim 130 are
used in (a).
133. The method of claim 125, wherein the DNA of (d) comprises a
library of DNA substrate molecules.
134. The method of claim 125, wherein the DNA substrate molecule
comprises a library of mutagenized DNA sequences encoding the
protein of interest.
135. The method of claim 125, wherein (d) further comprises
recovering DNA encoding the protein from affinity purified
particles of (c).
136. A method for optimizing secretion of a protein in a host by
evolving a gene encoding a secretory function, comprising: (a)
providing a cluster of genes encoding secretory functions; (b)
recombining at least a first and second sequence in the gene
cluster of (a) encoding a secretory function, the at least a first
and second sequences differing from each other in at least one
nucleotide, to generate a library of recombinant sequences; (c)
transforming a host cell culture with the products of (b), wherein
the host cell comprises a DNA sequence encoding the protein; (d)
subjecting the product of (c) to screening or selection for
secretion of the protein; and (e) recovering DNA encoding an
evolved gene encoding a secretory function from the product of
(d).
137. The method of claim 136, wherein the gene cluster comprises at
least one recognition site for a restriction endonuclease having
nonpalindromic ends at the cleavage site.
138. The method of claim 136, wherein the host is E. coli, yeast,
Bacillus, Pseudomonas, or a mammalian cell.
139. The method of claim 136, wherein the protein is a thermostable
DNA polymerase.
140. The method of claim 136, wherein protein is inducibly
expressed.
141. The method of claim 136, wherein the protein is linked to a
secretory leader sequence.
142. A secretory gene evolved by the method of claim 136.
143. The method of claim 136, wherein (a)-(e) are repeated.
144. The method of claim 136, wherein the DNA sequence of (c)
encodes all or part of a protein selected from Table I.
145. The method of claim 136, wherein the DNA sequence of (c)
comprises a library of mutant sequences.
146. The method of claim 136, wherein the products of (e) are
subjected to mutagenesis.
147. The method of claim 146, wherein mutagenesis comprises
recursive sequence recombination.
148. The method of claim 146, wherein the products of claim 146 are
used in (a).
149. The method of claim 136, wherein the DNA of (e) comprises a
library of evolved genes.
150. A method for evolving an improved DNA polymerase comprising:
(a) providing a library of mutant DNA substrate molecules encoding
mutant DNA polymerase; (b) screening extracts of cells transfected
with (a) and comparing activity with wild type DNA polymerase; (c)
recovering mutant DNA substrate molecules from cells in (b)
expressing mutant DNA polymerase having improved activity over
wild-type DNA polymerase; and (d) recovering a DNA substrate
molecule encoding an evolved polymerase from the products of
(c).
151. The method of claim 150, wherein the improved activity is at
least one of the group of higher quality sequencing ladder, less
termination of reactions with inosine, improve acceptance of base
analogs, improved acceptance of dideoxy nucleotides, and longer
sequencing ladders.
152. The method of claim 150, wherein the products of (a) are
expressed under control of arabinose promoter in an E. coli host
having a mutant host DNA polymerase.
153. The method of claim 150, wherein (a)-(d) are repeated.
154. An evolved DNA polymerase produced by the method of claim
150.
155. The method of claim 150, wherein the products of (d) are
subjected to mutagenesis.
156. The method of claim 155, wherein mutagenesis comprises
recursive sequence recombination.
157. The method of claim 155, wherein the products of claim 155 are
used in (a).
158. The method of claim 150, wherein the DNA substrate molecule of
(d) comprises a library of DNA substrate molecules.
159. A method for evolving a DNA polymerase with an error rate
greater than that of wild type DNA polymerase comprising: (a)
providing a library of mutant DNA substrate molecules encoding
mutant DNA polymerase in a host cell comprising an indicator gene
having a revertible mutation, wherein the indicator gene is
replicated by the mutant DNA polymerase; (b) screening the products
of (a) for revertants of the indicator gene; (c) recovering mutant
DNA substrate molecules from revertants; and (d) recovering a DNA
substrate molecule encoding an evolved polymerase from the products
of (c).
160. The method of claim 159, wherein the indicator gene is
LacZalpha or GFP.
161. The method of claim 159 wherein the revertible mutation is a
stop codon.
162. The method of claim 159, wherein the host cell comprises a
mutant host DNA polymerase.
163. A method for evolving a DNA polymerase, comprising: (a)
providing a library of mutant DNA substrate molecules encoding
mutant DNA polymerase, the library comprising a plasmid vector; (b)
preparing plasmid preparations and extracts of host cells
transfected with the products of (a); (c) amplifying each plasmid
preparation in a PCR reaction using the mutant polymerase encoded
by that plasmid, the polymerase being present in the host cell
extract; (d) recovering the PCR products of (c); and (e) recovering
a DNA substrate molecule encoding an evolved polymerase from the
products of (d).
164. The method of claim 163, wherein she reaction of (c) is
carried out in the presence of an organic solvent, a base analog,
or inosine.
165. The method of claim 163, wherein (a)-(e) are repeated.
166. An evolved polymerase produced by the method of claim 163.
167. The method of claim 163, wherein the products of (e) are
subjected to mutagenesis.
168. The method of claim 167, wherein mutagenesis comprises
recursive sequence recombination.
169. The method of claim 167, wherein the products of claim 167 are
used in (a).
170. The method of claim 163, wherein the DNA substrate molecule of
(e) comprises a library of DNA substrate molecules.
171. A method for evolving a p-nitrophenol phosphonatase from a
phosphonatase encoded by a DNA substrate molecule, comprising: (a)
providing library of mutants of the DNA substrate molecule, the
library comprising a plasmid expression vector; (b) transfecting a
host, wherein the host phn operon is deleted; (c) selecting or
growth of the transfectants of (b) using a p-nitrophenol
phosphonatase as a substrate; (d) recovering the DNA substrate
molecules from transfectants selected from (c); and (e) recovering
a DNA substrate molecule from (d) encoding an evolved
phosphonatase.
172. The method of claim 171, wherein (a)-(e) are repeated.
173. The method of claim 171, wherein the phosphonatase is selected
from the group consisting of beta-lactamase and alkyl
phosphonatase.
174. An evolved p-nitrophenol phosphonatase produced by the method
of claim 173.
175. The method of claim 171, wherein the products of (e) are
subjected to mutagenesis.
176. The method of claim 175, wherein mutagenesis comprises
recursive sequence recombination.
177. The method of claim 175, wherein the products of claim 175 are
used in (a).
178. The method of claim 171, wherein the DNA substrate molecule of
(e) comprises a library of DNA substrate molecules.
179. A method for evolving a protease encoded by a DNA substrate
molecule comprising: (a) providing library of mutants of the DNA
substrate molecule, the library comprising a plasmid expression
vector, wherein the DNA substrate molecule is linked to a secretory
leader; (b) transfecting a host; (c) selecting or growth of the
transfectants of (b) on a complex protein medium; and (d)
recovering a DNA substrate molecule from (c) encoding an evolved
protease.
180. The method of claim 179, wherein (a)-(d) are repeated.
181. An evolved subtilisin produced by the method of claim 179.
182. The method of claim 179, wherein the products of (d) are
subjected to mutagenesis.
183. The method of claim 182, wherein mutagenesis comprises
recursive sequence recombination.
184. The method of claim 182, wherein the products of claim 184 are
used in (a).
185. The method of claim 179, wherein the DNA substrate molecule of
(d) comprises a library of DNA substrate molecules.
186. The method of claim 179, wherein the protease is a
subtilisin.
187. A method for screening a library of protease mutants displayed
on a phage to obtain an improved protease, wherein a DNA substrate
molecule encoding the protease is fused to DNA encoding a
filamentous phage protein to generate a fusion protein, comprising:
(a) providing host cells expressing the fusion protein; (b)
overlaying host cells with a protein net to entrap the phage; (c)
washing the product of (b) to recover phage liberated by digestion
of the protein net; (d) recovering DNA from the product of (c); and
(e) recovering a DNA substrate from (d) encoding an improved
protease.
188. The method of claim 187, wherein (a)-(e) are repeated.
189. An evolved protease produced by the method of claim 187.
190. The method of claim 187, wherein the products of (e) are
subjected to mutagenesis.
191. The method of claim 190, wherein mutagenesis comprises
recursive sequence recombination.
192. The method of claim 190, wherein the products of claim 190 are
used in (a).
193. The method of claim 187, wherein the DNA substrate molecule of
(e) comprises a library of DNA substrate molecules.
194. A method for screening a library of protease mutants to obtain
an improved protease, the method comprising: (a) providing a
library of peptide substrates, the peptide substrate comprising a
fluorophore and a fluorescence quencher; (b) screening the library
of protease mutants for ability to cleave the peptide substrates,
wherein fluorescence is measured; and (c) recovering DNA encoding
at least one protease mutant from (b).
195. A method for evolving an alpha interferon gene comprising: (a)
providing a library of mutant alpha interferon genes, the library
comprising a filamentous phage vector; (b) stimulating cells
comprising a reporter construct, the reporter construct comprising
a reporter gene under control of an interferon responsive promoter,
and wherein the reporter gene is GFP; (c) separating the cells
expressing GFP by FACS; (d) recovering phage from the product of
(c); and (e) recovering an evolved interferon gene from the product
of (d).
196. The method of claim 195, wherein the interferon responsive
promoter is an MHC I promoter.
197. The method of claim 195, wherein (a)-(e) are repeated.
198. An evolved interferon produced by the method of claim 195.
199. The method of claim 195, wherein the products of (e) are
subjected to mutagenesis.
200. The method of claim 199, wherein mutagenesis comprises
recursive sequence recombination.
201. The method of claim 199, wherein the products of claim 199 are
used in (a).
202. The method of claim 195, wherein the evolved interferon gene
of (e) comprises a library of genes.
203. A method for screening a library of mutants of a DNA substrate
encoding a protein for an evolved DNA substrate, comprising: (a)
providing a library of mutants, the library comprising an
expression vector; (b) transfecting a mammalian host cell with the
library of (a), wherein mutant protein is expressed on the surface
of the cell; (c) screening or selecting the products of (b) with a
ligand for the protein; (d) recovering DNA encoding mutant protein
from the products of (c); and (e) recovering an evolved DNA
substrate from the products of (d).
204. The method of claim 203, wherein the ligand is an
antibody.
205. The method of claim 203, wherein the ligand is a substrate and
the protein is an enzyme.
206. The method of claim 203, wherein the expression vector
comprises an SV40 origin and the host cell is a Cos cell.
207. The method of claim 203, wherein the mutant protein is
expressed transiently.
208. The method of claim 203, wherein the host cell further
comprises SV40 large T antigen.
209. The method of claim 203, wherein the protein is an
antibody.
210. The method of claim 203, wherein (a)-(e) are repeated.
211. The method of claim 203, wherein the DNA substrate molecule
encodes all or part of a protein selected from Table I.
212. An evolved protein produced by the method of claim 203.
213. The method of claim 203, wherein the products of (e) are
subjected to mutagenesis.
214. The method of claim 213, wherein mutagenesis comprises
recursive sequence recombination.
215. The method of claim 213, wherein the products of claim 213 are
used in (a).
216. The method of claim 203, wherein the DNA substrate molecule of
(e) comprises a library of DNA substrate molecules.
217. A method for evolving a DNA substrate molecule encoding an
interferon alpha, comprising: (a) providing a library of mutant
alpha interferon genes, the library comprising an expression vector
wherein the alpha interferon genes are expressed under the control
of an inducible promoter; (b) transfecting host cells with the
library of (a); (c) contacting the product of (b) with a virus; (d)
recovering DNA encoding a mutant alpha interferon from host cells
surviving step (c); and (e) recovering an evolved interferon gene
from the product of (d).
218. The method of claim 217, wherein the promoter is a
metallothionein promoter.
219. The method of claim 217, wherein the virus is HIV.
220. The method of claim 217, wherein the virus further comprises a
conditionally lethal gene.
221. The method of claim 217, wherein the conditionally lethal gene
is thymidine kinase.
222. The method of claim 217, wherein the transfected cells are
exposed to conditionally lethal selective conditions.
223. The method of claim 217, wherein (a)-(e) are repeated.
224. An evolved IFN.alpha. polymerase produced by the method of
claim 217.
225. The method of claim 217, wherein the products of (e) are
subjected to mutagenesis.
226. The method of claim 225, wherein mutagenesis comprises
recursive sequence recombination.
227. The method of claim 225, wherein the products of claim 218 are
used in (a).
228. The method of claim 217, wherein the DNA substrate molecule of
(e) comprises a library of DNA substrate molecules.
229. A method for evolving the stability of a protein encoded by a
DNA substrate molecule, the DNA substrate molecule comprising a
fusion of a DNA sequence encoding the protein with a DNA sequence
encoding a filamentous phage protein to generate a fusion protein,
the method comprising: (a) providing a host cell expressing a
library of mutants of the fusion protein; (b) affinity purifying
the mutants with a ligand for the protein, wherein the ligand is a
human serum protein, tissue specific protein, or receptor; (c)
recovering DNA encoding a mutant protein from the affinity selected
mutants of (b); and (d) recovering an evolved gene encoding the
protein from the product of (c).
230. The method of claim 229, wherein the serum protein is serum
albumin, immunoglobulin, lipoprotein, haptoglobin, fibrinogen,
transferrin, alpha-1 anti-trypsin, or alpha -2 macroglobulin.
231. The method of claim 229, wherein the DNA sequence encoding the
filamentous phage protein comprises a phage.
232. The method of claim 229, wherein the DNA sequence encoding the
filamentous phage protein comprises a phagemid.
233. The method of claim 229, wherein the products of step (a) are
derivitized with a half-life extending moiety.
234. The method of claim 229, wherein the moiety is polyethylene
glycol.
235. The method of claim 229, wherein the DNA substrate molecule
comprises a fusion of nucleic acid encoding the protein with
nucleic acid encoding an epitope tag.
236. The method of claim 235, wherein the products of (a) are
contacted with a protease prior to (b).
237. The method of claim 235, wherein the ligand is an antibody
specific for the epitope tag.
238. The method of claim 229, wherein the protein is selected from
Table I.
239. The method of claim 229, wherein the products of (a) are
subjected to heat, metal ions, non-physiological pH,
lyophilization, or freeze-thawing before (b).
240. The method of claim 229, wherein (a)-(e) are repeated.
241. An evolved polymerase produced by the method of claim 229.
242. The method of claim 229, wherein the products of (d) are
subjected to mutagenesis.
243. The method of claim 242, wherein mutagenesis comprises
recursive sequence recombination.
244. The method of claim 242, wherein the products of claim 242 are
used in (a).
245. The method of claim 229, wherein the evolved gene of (d)
comprises a library of DNA substrate molecules.
246. A method for evolving a protein having at least two subunits,
comprising: (a) providing a library of mutant DNA substrate
molecules for each subunit; (b) recombining the libraries into a
library of single chain constructs of the protein, the single chain
construct comprising a DNA substrate molecule encoding each subunit
sequence, the subunit sequence being linked by a linker at a
nucleic acid sequence encoding the amino terminus of one subunit to
a nucleic acid sequence encoding the carboxy terminus of a second
subunit; (c) screening or selecting the products of (b), (d)
recovering recombinant single chain construct DNA substrate
molecules from the products of (c); (e) subjecting the products of
(d) to mutagenesis; and (f) recovering an evolved single chain
construct DNA substrate molecule from (e).
247. The method of claim 246, wherein the products of (b) are
displayed on a phage.
248. The method of claim 246, wherein the protein is selected from
Table I.
249. The method of claim 246, wherein (a)-(f) are repeated.
250. An evolved protein produced by the method of claim 246.
251. The method of claim 246, wherein the products of (f) are
subjected to mutagenesis.
252. The method of claim 246, wherein mutagenesis comprises
recursive sequence recombination.
253. The method of claim 246, wherein the products of claim 246 are
used in (a).
254. The method of claim 246, wherein the evolved DNA substrate
molecule of (f) comprises a library of DNA substrate molecules.
255. A method for evolving the coupling of a mammalian
7-transmembrane receptor to a yeast signal transduction pathway,
comprising: (a) expressing a library of mammalian G alpha protein
mutants in a host yeast cell, wherein the host cell expresses the
mammalian 7-transmembrane receptor and a reporter gene, the
receptor gene geing expressed under control of a yeast pheromone
responsive promoter; (b) screening or selecting the products of (a)
for expression of the reporter gene in the presence of a ligand for
the 7-transmembrance receptor; and (c) recovering DNA encoding an
evolved G alpha protein mutant from screened or selected products
of (b).
256. The method of claim 255, wherein the products of (c) are
subjected to mutagenesis.
257. The method of claim 256, wherein mutagenesis comprises
recursive sequence recombination.
258. The method of claim 255, wherein the products of claim 255 are
used in (a).
259. The method of claim 255, wherein (a)-(c) are repeated.
260. An evolved G alpha protein produced by the method of claim
255.
261. The method of claim 255, wherein the reporter gene is
luciferase.
262. The method of claim 255, wherein the pheromone responsive
promoter is positively regulated by GAL4 and wherein GAL4 is
expressed under the control of a pheromone sensitive, GAL4 enhanced
promoter.
263. A method for recombining at least a first and second DNA
substrate molecule, comprising: (a) transfecting a host cell with
at least a first and second DNA substrate molecule wherein the at
least a first and second DNA substrate molecules are recombined in
the host cell; (b) screening or selecting the products of (a) for a
desired property; and (c) recovering recombinant DNA substrate
molecules from (b).
264. The method of claim 263, wherein the products of (c) are
subjected to mutagenesis.
265. The method of claim 264, wherein the mutagenesis comprises
recursive sequence recombination.
266. The method of claim 263, wherein (a)-(c) are repeated.
267. The method of claim 263, wherein the products of claim 263 are
used in (a).
268. A method for evolving a DNA substrate sequence encoding a
protein of interest, wherein the DNA substrate comprises a vector,
he vector comprising single-stranded DNA, the method comprising:
(a) providing single-stranded vector DNA and a library of mutants
of the DNA substrate sequence; (b) annealing denatured
double-stranded DNA from the library of (a) to the single stranded
vector DNA of (a); (c) transforming the products of (b) into a
host; (d) screening the product of (c) for a desired property; and
(e) recovering evolved DNA substrate DNA from the products of
(d).
269. The method of claim 268, wherein the product of (e) is
subjected to mutagenesis.
270. The method of claim 269, wherein mutagenesis comprises
recursive sequence recombination.
271. The method of claim 269, wherein the product of claim 269 is
used in (a).
272. The method of claim 268, wherein the host is a mutS host.
273. The method of claim 268, wherein the vector is a phagemid.
Description
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 08/198,431, filed Feb. 17, 1994, Ser. No.
PCT/US95/02126, filed, Feb. 17, 1995, Ser. No. 08/425,684, filed
Apr. 18, 1995, Ser. No. 08/537,874, filed Oct. 30, 1995, Ser. No.
08/564,955, filed Nov. 30, 1995, Ser. No. 08/621,859, filed Mar.
25, 1996, Ser. No. 08/621,430, filed Mar. 25, 1996, Ser. No.
PCT/US96/05480, filed Apr. 18, 1996, Ser. No. 08/650,400, filed May
20, 1996, Ser. No. 08/675,502, filed Jul. 3, 1996, Ser. No.
08/721,824, filed Sep. 27, 1996, and Ser. No. 08/722,660 filed Sep.
27, 1996 the specifications of which are herein incorporated by
reference in their entirety for all purposes.
BACKGROUND OF THE INVENTION
[0002] Recursive sequence recombination entails performing
iterative cycles of recombination and screening or selection to
"evolve" individual genes, whole plasmids or viruses, multigene
clusters, or even whole genomes (Stemmer, Bio/Technology 13:549-553
(1995)). Such techniques do not require the extensive analysis and
computation required by conventional methods for polypeptide
engineering. Recursive sequence recombination allows the
recombination of large numbers of mutations in a minimum number of
selection cycles, in contrast to traditional, pairwise
recombination events.
[0003] Thus, recursive sequence recombination (RSR) techniques
provide particular advantages in that they provide recombination
between mutations in any or all of these, thereby providing a very
fast way of exploring the manner in which different combinations of
mutations can affect a desired result.
[0004] In some instances, however, structural and/or functional
information is available which although not required for recursive
sequence recombination, provides opportunities for modification of
the technique. In other instances, selection and/or screening of a
large number of recombinants can be costly or time-consuming. A
further problem can be the manipulation of large nucleic acid
molecules. The instant invention addresses these issues and
others.
SUMMARY OF THE INVENTION
[0005] One aspect of the invention is a method for evolving a
protein encoded by a DNA substrate molecule comprising:
[0006] (a) digesting at least a first and second DNA substrate
molecule, wherein the at least a first and second substrate
molecules differ from each other in at least one nucleotide, with a
restriction endonuclease;
[0007] (b) ligating the mixture to generate a library of
recombinant DNA molecules;
[0008] (c) screening or selecting the products of (b) for a desired
property; and
[0009] (d) recovering a recombinant DNA substrate molecule encoding
an evolved protein.
[0010] A further aspect of the invention is a method for evolving a
protein encoded by a DNA substrate molecule by recombining at least
a first and second DNA substrate molecule, wherein the at least a
first and second substrate molecules differ from each other in at
least one nucleotide and comprise defined segments, the method
comprising:
[0011] (a) providing a set of oligonucleotide PCR primers,
comprising at least one primer for each segment, wherein the primer
sequence is complementary to at least one junction with another
segment;
[0012] (b) amplifying the segments of the at least a first and
second DNA substrate molecules with the primers of step (a) in a
polymerase chain reaction;
[0013] (c) assembling the products of step (b) to generate a
library of recombinant DNA substrate molecules;
[0014] (d) screening or selecting the produces of (c) for a desired
property; and
[0015] (e) recovering a recombinant DNA substrate molecule from (d)
encoding an evolved protein.
[0016] A further aspect of the invention is a method of enriching a
population of DNA fragments for mutant sequences comprising:
[0017] (a) denaturing and renaturing the population of fragments to
generate a population of hybrid double-stranded fragments in which
at least one double-stranded fragment comprises at least one base
pair mismatch;
[0018] (b) fragmenting the products of (a) into fragments of about
20-100 bp;
[0019] (c) affinity-purifying fragments having a mismatch on an
affinity matrix to generate a pool of DNA fragments enriched for
mutant sequences; and
[0020] (d) assembling the products of (c) to generate a library of
recombinant DNA substrate molecules.
[0021] A further aspect of the invention is a method for evolving a
protein encoded by a DNA substrate molecule, by recombining at
least a first and second DNA substrate molecule, wherein the at
least a first and second substrate molecules share a region of
sequence homology of about 10 to 100 base pairs and comprise
defined segments, the method comprising:
[0022] (a) providing regions of homology in the at least a first
and second DNA substrate molecules by inserting an intron sequence
between at least two defined segments;
[0023] (b) fragmenting and recombining DNA substrate molecules of
(a), wherein regions of homology are provided by the introns;
[0024] (c) screening or selecting the products of (b) for a desired
property; and
[0025] (d) recovering a recombinant DNA substrate molecule from the
products of (c) encoding an evolved protein.
[0026] A further aspect of the invention is a method for evolving a
protein encoded by a DNA substrate molecule by recombining at least
a first and second DNA substrate molecule, wherein the at least a
first and second substrate molecules differ from each other in at
east one nucleotide and comprise defined segments, the method
comprising:
[0027] (a) providing a set of oligonucleotide PCR primers, wherein
for each strand of each segment a pair of primers is provided, one
member of each pair bridging the junction at one end of the segment
and the other bridging the junction at the other end of the
segment, with the terminal ends of the DNA molecule having as one
member of the pair a generic primer, and wherein a set of primers
is provided for each of the at least a first and second substrate
molecules;
[0028] (b) amplifying the segments of the at least a first and
second DNA substrate molecules with the primers of (a) in a
polymerase chain reaction;
[0029] (c) assembling the products of (b) to generate a pool of
recombinant DNA molecules;
[0030] (d) selecting or screening the products of (c) for a desired
property; and
[0031] (e) recovering a recombinant DNA substrate molecule from the
products of (d) encoding an evolved protein.
[0032] A further aspect of the invention is a method for optimizing
expression of a protein by evolving the protein, wherein the
protein is encoded by a DNA substrate molecule, comprising:
[0033] (a) providing a set of oligonucleotides, wherein each
oligonucleotide comprises at least two regions complementary to the
DNA molecule and at least one degenerate region, each degenerate
region encoding a region of an amino acid sequence of the
protein;
[0034] (b) assembling the set of oligonucleotides into a library of
full length genes;
[0035] (c) expressing the products of (b) in a host cell;
[0036] (d) screening the products of (c) for improved expression of
the protein; and
[0037] (e) recovering a recombinant DNA substrate molecule encoding
an evolved protein from (d).
[0038] A further aspect of the invention is a method for optimizing
expression of a protein encoded by a DNA substrate molecule by
evolving the protein, wherein the DNA substrate molecule comprises
at least one lac operator and a fusion of a DNA sequence encoding
the protein with a DNA sequence encoding a lac headpiece dimer, the
method comprising:
[0039] (a) transforming a host cell with a library of mutagenized
DNA substrate molecules;
[0040] (b) inducing expression of the protein encoded by the
library of (a);
[0041] (c) preparing an extract of the product of (b);
[0042] (d) fractionating insoluble protein from complexes of
soluble protein and DNA; and
[0043] (e) recovering a DNA substrate molecule encoding an evolved
protein from (d).
[0044] A further aspect of the invention is a method for evolving
functional expression of a protein encoded by a DNA substrate
molecule comprising a fusion of a DNA sequence encoding the protein
with a DNA sequence encoding filamentous phage protein to generate
a fusion protein, the method comprising:
[0045] (a) providing a host cell producing infectious particles
expressing a fusion protein encoded by a library of mutagenized DNA
substrate molecules;
[0046] (b) recovering from (a) infectious particles displaying the
fusion protein;
[0047] (c) affinity purifying particles displaying the mutant
protein using a ligand for the protein; and
[0048] (d) recovering a DNA substrate molecule encoding an evolved
protein from affinity purified particles of (c).
[0049] A further aspect of the invention is a method for optimizing
expression of a protein encoded by a DNA substrate molecule
comprising a fusion of a DNA sequence encoding the protein with a
lac headpiece dimer, wherein the DNA substrate molecule is present
on a first plasmid vector, the method comprising:
[0050] (a) providing a host cell transformed with the first vector
and a second vector comprising a library of mutants of at least one
chaperonin gene, and at least one lac operator;
[0051] (b) preparing an extract of the product of (a);
[0052] (c) fractionating insoluble protein from complexes of
soluble protein and DNA; and
[0053] (d) recovering DNA encoding a chaperonin gene from (c).
[0054] A further aspect of the invention is a method for optimizing
expression of a protein encoded by a DNA substrate molecule
comprising a fusion of a DNA sequence encoding the protein with a
filamentous phage gene, wherein the fusion is carried on a phagemid
comprising a library of chaperonin gene mutants, the method
comprising:
[0055] (a) providing a host cell producing infectious particles
expressing a fusion protein encoded by a library of mutagenized DNA
substrate molecules;
[0056] (b) recovering from (a) infectious particles displaying the
fusion protein;
[0057] (c) affinity purifying particles displaying the protein
using a ligand for the protein; and
[0058] (d) recovering DNA encoding the mutant chaperonin from
affinity purified particles of (c).
[0059] A further aspect of the invention is a method for optimizing
secretion of a protein in a host by evolving a gene encoding a
secretory function, comprising:
[0060] (a) providing a cluster of genes encoding secretory
functions;
[0061] (b) recombining at least a first and second sequence in the
gene cluster of (a) encoding a secretory function, the at least a
first and second sequences differing from each other in at least
one nucleotide, to generate a library of recombinant sequences;
[0062] (c) transforming a host cell culture with the products of
(b), wherein the host cell comprises a DNA sequence encoding the
protein;
[0063] (d) subjecting the product of (c) to screening or selection
for secretion of the protein; and
[0064] (e) recovering DNA encoding an evolved gene encoding a
secretory function from one product of (d).
[0065] A further aspect of the invention is a method for evolving
an improved DNA polymerase comprising:
[0066] (a) providing a library of mutant DNA substrate molecules
encoding mutant DNA polymerase;
[0067] (b) screening extracts of cells transfected with (a) and
comparing activity with wild type DNA polymerase;
[0068] (c) recovering mutant DNA substrate molecules from cells in
(b) expressing mutant DNA polymerase having improved activity over
wild-type DNA polymerase; and
[0069] (d) recovering a DNA substrate molecule encoding an evolved
polymerase from the products of (c).
[0070] A further aspect of the invention is a method for evolving a
DNA polymerase with an error rate greater than that of wild type
DNA polymerase comprising:
[0071] (a) providing a library of mutant DNA substrate molecules
encoding mutant DNA polymerase in a host cell comprising an
indicator gene having a revertible mutation, wherein the indicator
gene is replicated by the mutant DNA polymerase;
[0072] (b) screening the products of (a) for revertants of the
indicator gene;
[0073] (c) recovering mutant DNA substrate molecules from
revertants; and
[0074] (d) recovering a DNA substrate molecule encoding an evolved
polymerase from the products of (c).
[0075] A further aspect of the invention is a method for evolving a
DNA polymerase, comprising:
[0076] (a) providing a library of mutant DNA substrate molecules
encoding mutant DNA polymerase, the library comprising a plasmid
vector;
[0077] (b) preparing plasmid preparations and extracts of host
cells transfected with the products of (a);
[0078] (c) amplifying each plasmid preparation in a PCR reaction
using the mutant polymerase encoded by that plasmid, the polymerase
being present in the host cell extract;
[0079] (d) recovering the PCR produces of (c); and
[0080] (e) recovering a DNA substrate molecule encoding an evolved
polymerase from products of (d).
[0081] A further aspect of the invention is a method for evolving a
p-nitrophenol phosphonatase from a phosphonatase encoded by a DNA
substrate molecule, comprising:
[0082] (a) providing library of mutants of the DNA substrate
molecule, the library comprising a plasmid expression vector;
[0083] (b) transfecting a host, wherein the host phn operon is
deleted;
[0084] (c) selecting for growth of the transfectants of (b) using a
p-nitrophenol phosphonatase as a substrate;
[0085] (d) recovering the DNA substrate molecules from
transfectants selected from (c); and
[0086] (e) recovering a DNA substrate molecule from (d) encoding an
evolved phosphonatase.
[0087] A further aspect of the invention is a method for evolving a
protease encoded by a DNA substrate molecule comprising:
[0088] (a) providing library of mutants of the DNA substrate
molecule, the library comprising a plasmid expression vector,
wherein the DNA substrate molecule is linked to a secretory
leader;
[0089] (b) transfecting a host;
[0090] (c) selecting for growth of the transfectants of (b) on a
complex protein medium; and
[0091] (d) recovering a DNA substrate molecule from (c) encoding an
evolved protease.
[0092] A further aspect of the invention is a method for screening
a library of protease mutants displayed on a phage to obtain an
improved protease, wherein a DNA substrate molecule encoding the
protease is fused to DNA encoding a filamentous phage protein to
generate a fusion protein, comprising:
[0093] (a) providing host cells expressing the fusion protein;
[0094] (b) overlaying host cells with a protein net to entrap the
phage;
[0095] (c) washing the product of (b) to recover phage liberated by
digestion of the protein net;
[0096] (d) recovering DNA from the product of (c); and
[0097] (e) recovering a DNA substrate from (d) encoding an improved
protease.
[0098] A further aspect of the invention is a method for screening
a library of protease mutants to obtain an improved protease, the
method comprising:
[0099] (a) providing a library of peptide substrates, the peptide
substrate comprising a fluorophore and a fluorescence quencher;
[0100] (b) screening the library of protease mutants for ability to
cleave the peptide substrates, wherein fluorescence is measured;
and
[0101] (c) recovering DNA encoding at least one protease mutant
from (b).
[0102] A further aspect of the invention is a method for evolving
an alpha interferon gene comprising:
[0103] (a) providing a library of mutant alpha interferon genes,
the library comprising a filamentous phage vector;
[0104] (b) stimulating cells comprising a reporter construct, the
reporter construct comprising a reporter gene under control of an
interferon responsive promoter, and wherein the reporter gene is
GFP;
[0105] (c) separating the cells expressing GFP by FACS;
[0106] (d) recovering phage from the product of (c); and
[0107] (e) recovering an evolved interferon gene from the product
of (d).
[0108] A further aspect of the invention is a method for screening
a library of mutants of a DNA substrate encoding a protein for an
evolved DNA substrate, comprising:
[0109] (a) providing a library of mutants, the library comprising
an expression vector;
[0110] (b) transfecting a mammalian host cell with the library of
(a), wherein mutant protein is expressed on the surface of the
cell;
[0111] (c) screening or selecting the products of (b) with a ligand
for the protein;
[0112] (d) recovering DNA encoding mutant protein from the products
of (c); and
[0113] (e) recovering an evolved DNA substrate from the products of
(d).
[0114] A further aspect of the invention is a method for evolving a
DNA substrate molecule encoding an interferon alpha,
comprising:
[0115] (a) providing a library of mutant alpha interferon genes,
the library comprising an expression vector wherein the alpha
interferon genes are expressed under the control of an inducible
promoter;
[0116] (b) transfecting host cells with the library of (a);
[0117] (c) contacting the product of (b) with a virus;
[0118] (d) recovering DNA encoding a mutant alpha interferon from
host cells surviving step (c); and
[0119] (e) recovering an evolved interferon gene from the product
of (d).
[0120] A further aspect of the invention is a method for evolving
the stability of a protein encoded by a DNA substrate molecule, the
DNA substrate molecule comprising a fusion of a DNA sequence
encoding the protein with a DNA sequence encoding a filamentous
phage protein to generate a fusion protein, the method
comprising:
[0121] (a) providing a host cell expressing a library of mutants of
the fusion protein;
[0122] (b) affinity purifying the mutants with a ligand for the
protein, wherein the ligand is a human serum protein, tissue
specific protein, or receptor;
[0123] (c) recovering DNA encoding a mutant protein from the
affinity selected mutants of (b); and
[0124] (d) recovering an evolved gene encoding the protein from the
product of (c).
[0125] A further aspect of the invention is a method for evolving a
protein having at least two subunits, comprising:
[0126] (a) providing a library of mutant DNA substrate molecules
for each subunit;
[0127] (b) recombining the libraries into a library of single chain
constructs of the protein, the single chain construct comprising a
DNA substrate molecule encoding each subunit sequence, the subunit
sequence being linked by a linker at a nucleic acid sequence
encoding the amino terminus of one subunit to a nucleic acid
sequence encoding the carboxy terminus of a second subunit;
[0128] (c) screening or selecting the products of (B),
[0129] (d) recovering recombinant single chain construct DNA
substrate molecules from the products of (c);
[0130] (e) subjecting the products of (d) to mutagenesis; and
[0131] (f) recovering an evolved single chain construct DNA
substrate molecule from (e).
[0132] A further aspect of the invention is a method for evolving
the coupling of a mammalian 7-transmembrane receptor to a yeast
signal transduction pathway, comprising:
[0133] (a) expressing a library of mammalian G alpha protein
mutants in a host cell, wherein the host cell expresses the
mammalian 7-transmembrane receptor and a reporter gene, the
receptor gene geing expressed under control of a pheromone
responsive promoter;
[0134] (b) screening or selecting the products of (a) for
expression of the reporter gene in the presence of a ligand for the
7-transmembrance receptor; and
[0135] (c) recovering DNA encoding an evolved G alpha protein
mutant from screened or selected products of (b).
[0136] A further aspect of the invention is a method for
recombining at least a first and second DNA substrate molecule,
comprising:
[0137] (a) transfecting a host cell with at least a first and
second DNA substrate molecule wherein the at least a first and
second DNA substrate molecules are recombined in the host cell;
[0138] (b) screening or selecting the products of (a) for a desired
property; and
[0139] (c) recovering recombinant DNA substrate molecules from
(b).
[0140] A further aspect of the invention is a method for evolving a
DNA substrate sequence encoding a protein of interest, wherein the
DNA substrate comprises a vector, the vector comprising
single-stranded DNA, the method comprising:
[0141] (a) providing single-stranded vector DNA and a library of
mutants of the DNA substrate sequence;
[0142] (b) annealing single stranded DNA from the library of (a) to
the single stranded vector DNA of (a);
[0143] (c) transforming the products of (b) into a host;
[0144] (d) screening the product of (c) for a desired property;
and
[0145] (e) recovering evolved DNA substrate DNA from the products
of (d).
BRIEF DESCRIPTION OF THE DRAWINGS
[0146] FIG. 1 depicts the alignment of oligo PCR primers for
evolution of bovine calf intestinal alkaline phosphatase.
[0147] FIG. 2 depicts the alignment of alpha interferon amino acid
and nucleic acid sequences.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0148] The invention provides a number of strategies for evolving
polypeptides through recursive recombination methods. In some
embodiments, the strategies of the invention can generally be
classified as "coarse grain shuffling" and "fine grain shuffling."
As described in detail below, these strategies are especially
applicable in situations where some structural or functional
information is available regarding the polypeptides of interest,
where the nucleic acid to be manipulated is large, when selection
or screening of many recombinants is cumbersome, and so on. "Coarse
grain shuffling" generally involves the exchange or recombination
of segments of nucleic acids, whether defined as functional
domains, exons, restriction endonuclease fragments, or otherwise
arbitrarily defined segments. "Fine grain shuffling" generally
involves the introduction of sequence variation within a segment,
such as within codons.
[0149] Coarse grain and fine grain shuffling allow analysis of
variation occuring within a nucleic acid sequence, also termed
"searching of sequence space." Although both techniques are
meritorious, the results are qualitatively different. For example,
coarse grain searches are often better suited for optimizing
multigene clusters such as polyketide operons, whereas fine grain
searches are often optimal for optimizing a property such as
protein expression using codon usage libraries.
[0150] The strategies generally entail evolution of gene(s) or
segment(s) thereof to allow retention of function in a heterologous
cell or improvement of function in a homologous or heterologous
cell. Evolution is effected generally by a process termed recursive
sequence recombination. Recursive sequence recombination can be
achieved in many different formats and permutations of formats, as
described in further detail below. These formats share some common
principles. Recursive sequence recombination entails successive
cycles of recombination to generate molecular diversity, i.e., the
creation of a family of nucleic acid molecules showing substantial
sequence identity to each other but differing in the presence of
mutations. Each recombination cycle is followed by at least one
cycle of screening or selection for molecules having a desired
characteristic. The molecule(s) selected in one round form the
starting materials for generating diversity in the next round. In
any given cycle, recombination can occur in vivo or in vitro.
Furthermore, diversity resulting from recombination can be
augmented in any cycle by applying prior methods of mutagenesis
(e.g., error-prone PCR or cassette mutagenesis, passage through
bacterial mutator strains, treatment with chemical mutagens) to
either the substrates for or products of recombination.
[0151] I. Formats for Recursive Sequence Recombination
[0152] Some formats and examples for recursive sequence
recombination, sometimes referred to as DNA shuffling, evolution,
or molecular breeding, have been described by the present inventors
and co-workers in co-pending applications U.S. patent application
Ser. No. 08/198,431, filed Feb. 17, 1994, Ser. No. PCT/US95/02126,
filed, Feb. 17, 1995, Ser. No. 08/425,684, filed Apr. 8, 1995, Ser.
No. 08/537,874, filed Oct. 30, 1995, Ser. No. 08/564,955, filed
Nov. 30, 1995, Ser. No. 38/621,859, filed Mar. 25, 1996, Ser. No.
08/621,430, filed Mar. 25, 1996, Ser. No. PCT/US96/05480, filed
Apr. 18, 1996, Ser. No. 08/650,400, filed May 20, 1996, Ser. No.
08/675,502, filed Jul. 3, 1996, Ser. No. 08/721, 824, filed Sep.
27, 1996, and Ser. No. 08/722,660 filed Sep. 27, 1996; Stemmer,
Science 270:1510 (1995); Stemmer et al., Gene 164:49-53 (1995);
Stemmer, Bio/Technology 13:549-553 (1995); Stemmer, Proc. Natl.
Acad. Sci. U.S.A. 91:10747-10751 (1994); Stemmer, Nature
370:389-391 (1994); Crameri et al., Nature Medicine 2(1):1-3
(1996); Crameri et al., Nature Biotechnology 14:315-319 (1996),
each of which is incorporated by reference in its entirety for all
purposes.
[0153] In general, the term "gene" is used herein broadly to refer
to any segment or sequence of DNA associated with a biological
function. Genes can be obtained from a variety of sources,
including cloning from a source of interest or synthesizing from
known or predicted sequence information, and may include sequences
designed to have desired parameters.
[0154] A wide variety of cell types can be used as a recipient of
evolved genes. Cells of particular interest include many bacterial
cell types, both gram-negative and gram-positive, such as
Rhodococcus, Streptomycetes, Actinomycetes, Corynebacteria,
Penicillium, Bacillus, Escherichia coli, Pseudomonas, Salmonella,
and Erwinia. Cells of interest also include eukaryotic cells,
particularly mammalian cells (e.g., mouse, hamster, primate,
human), both cell lines and primary cultures. Such cells include
stem cells, including embryonic stem cells, zygotes, fibroblasts,
lymphocytes, Chinese hamster ovary CHO, mouse fibroblasts (NIH3T3),
kidney, liver, muscle, and skin cells. Other eukaryotic cells of
interest include plant cells, such as maize, rice, wheat, cotton,
soybean, sugarcane, tobacco, and arabidopsis; fish, algae, fungi,
Penicillium, Fusarium, Aspergillus, Podospora, Neurospora),
insects, yeasts (Picchia and Saccharomyces).
[0155] The choice of host will depend on a number of factors,
depending on the intended use of the engineered host, including
pathogenicity, substrate range, environmental hardiness, presence
of key intermediates, ease of genetic manipulation, and likelihood
of promiscuous transfer of genetic information to other organisms.
A preferred host has the ability to replicate vector DNA, express
proteins of interest, and properly traffic proteins of interest.
Particularly advantageous hosts are E. coli, lactobacilli,
Streptomycetes, Actinomycetes, fungi such as Saccaromyces
cerivisiae or Pischia pastoris, Schneider cells, L-cells, COS
cells, CHO cells, and transformed B cell lines such as SP2/0, J558,
NS-1 and AG8-653.
[0156] The breeding procedure starts with at least two substrates
that generally show substantial sequence identity to each other
(i.e., at least about 50%, 70%, 80% or 90% sequence identity), but
differ from each other at certain positions. The difference can be
any type of mutation, for example, substitutions, insertions and
deletions. Often, different segments differ from each other in
perhaps 5-20 positions. For recombination to generate increased
diversity relative to the starting materials, the starting
materials must differ from each other in at least two nucleotide
positions. That is, if there are only two substrates, there should
be at least two divergent positions. If there are three substrates,
for example, one substrate can differ from the second as a single
position, and the second can differ from the third at a different
single position. The starting DNA segments can be natural variants
of each other, for example, allelic or species variants. The
segments can also be from nonallelic genes showing some degree of
structural and usually functional relatedness e.g., different genes
within a superfamily such as the immunoglobulin superfamily). The
starting DNA segments can also be induced variants of each other.
For example, one DNA segment can be produced by error-prone PCR
replication of the other, or by substitution of a mutagenic
cassette. Induced mutants can also be prepared by propagating one
(or both) of the segments in a mutagenic strain. In these
situations, strictly speaking, the second DNA segment is not a
single segment but a large family of related segments. The
different segments forming the starting materials are often the
same length or substantially the same length. However, this need
not be the case; for example; one segment can be a subsequence of
another. The segments can be present as part of larger molecules,
such as vectors, or can be in isolated form.
[0157] The starting DNA segments are recombined by any of the
recursive sequence recombination formats provded herein to generate
a diverse library of recombinant DNA segments. Such a library can
vary widely in size from having fewer than 10 to more than
10.sup.5, 10.sup.9, or 10.sup.12 members. In general, the starting
segments and the recombinant libraries generated include
full-length coding sequences and any essential regulatory
sequences, such as a promoter and polyadenylation sequence,
required for expression. However, if this is not the case, the
recombinant DNA segments in the library can be inserted into a
common vector providing the missing sequences before performing
screening/selection.
[0158] If the recursive sequence recombination format employed is
an in vivo format, the library of recombinant DNA segments
generated already exists in a cell, which is usually the cell type
in which expression of the enzyme with altered substrate
specificity is desired. If recursive sequence recombination is
performed in vitro, the recombinant library is preferably
introduced into the desired cell type before screening/selection.
The members of the recombinant library can be linked to an episome
or virus before introduction or can be introduced directly. In some
embodiments of the invention, the library is amplified in a first
host, and is then recovered from that host and introduced to a
second host more amenable to expression, selection, or screening,
or any other desirable parameter. The manner in which the library
is introduced into the cell type depends on the DNA-uptake
characteristics of the cell type, e.g., having viral receptors,
being capable of conjugation, or being naturally competent. If the
cell type is insusceptible to natural and chemical-induced
competence, but susceptible to electroporation, one would usually
employ electroporation. If the cell type is insusceptible to
electroporation as well, one can employ biolistics. The biolistic
PDS-1000 Gene Gun (Biorad, Hercules, Calif.) uses helium pressure
to accelerate DNA-coated gold or tungsten microcarriers toward
target cells. The process is applicable to a wide range of tissues,
including plants, bacteria, fungi, algae, intact animal tissues,
tissue culture cells, and animal embryos. One can employ electronic
pulse delivery, which is essentially a mild electroporation format
for live tissues in animals and patients. Zhao, Advanced Drug
Delivery Reviews 17:257-262 (1995). Novel methods for making cells
competent are described in co-pending application U.S. patent
application Ser. No. 08/621,430, filed Mar. 25, 1996. After
introduction of the library of recombinant DNA genes, the cells are
optionally propagated to allow expression of genes to occur.
[0159] A. In Vitro Formats
[0160] One format for recursive sequence recombination utilizes a
pool of related sequences. The sequences can be DNA or RNA and can
be of various lengths depending on the size of the gene or DNA
fragment to be recombined or reassembled. Preferably the sequences
are from 50 bp to 100 kb.
[0161] The pool of related substrates can be fragmented, usually at
random, into fragments of from about 5 bp to 5 kb or more.
Preferably the size of the random fragments is from about 10 bp to
1000 bp, more preferably the size of the DNA fragments is from
about 20 bp to 500 bp. The substrates can be digested by a number
of different methods, such as DNAseI or RNAse digestion, random
shearing or restriction enzyme digestion. The concentration of
nucleic acid fragments of a particular length is often less than
0.1% or 1% by weight of the total nucleic acid. The number of
different specific nucleic acid fragments in the mixture is usually
at least about 100, 500 or 1000.
[0162] The mixed population of nucleic acid fragments are denatured
by heating to about 80.degree. C. to 100.degree. C., more
preferably from 90.degree. C. to 96.degree. C., to form
single-stranded nucleic acid fragments. Single-stranded nucleic
acid fragments having regions of sequence identity with other
single-stranded nucleic acid fragments can then be reannealed by
cooling to 20.degree. C. to 75.degree. C., and preferably from
40.degree. C. to 65.degree. C. Renaturation can be accelerated by
the addition of polyethylene glycol ("PEG") or salt. The salt
concentration is preferably from 0 mM to 600 mM, more preferably
the salt concentration is from 10 mM to 100 mM. The salt may be
such salts as (NH.sub.4).sub.2SO.sub.4, KCl, or NaCl. The
concentration of PEG is preferably from 0% to 20%, more preferably
from 5% to 10%. The fragments that reanneal can be from different
substrates.
[0163] The annealed nucleic acid fragments are incubated in the
presence of a nucleic acid polymerase, such as Taq or Klenow,
Mg.sup.++ at 1 mM-20 mM, and dNTP's (i.e. dATP, dCTP, dGTP and
dTTP). If regions of sequence identity are large, Taq or other
high-temperature polymerase can be used with an annealing
temperature of between 45-65.degree. C. If the areas of identity
are small, Klenow or other low-temperature polymerases can be used
with an annealing temperature of between 20-30.degree. C. The
polymerase can be added to the random nucleic acid fragments prior
to annealing, simultaneously with annealing or after annealing.
[0164] The cycle of denaturation, renaturation and incubation of
random nucleic acid fragments in the presence of polymerase is
sometimes referred to as "shuffling" of the nucleic acid in vitro.
This cycle is repeated for a desired number of times. Preferably
the cycle is repeated from 2 to 100 times, more preferably the
sequence is repeated from 10 to 40 times. The resulting nucleic
acids are a family of double-stranded polynucleotides of from about
50 bp to about 100 kb, preferably from 500 bp to 50 kb. The
population represents variants of the starting substrates showing
substantial sequence identity thereto but also diverging at several
positions. The population has many more members than the starting
substrates. The population of fragments resulting from
recombination is preferably first amplified by PCR, then cloned
into an appropriate vector and the ligation mixture used to
transform host cells.
[0165] In a variation of in vitro shuffling, subsequences of
recombination substrates can be generated by amplifying the
full-length sequences under conditions which produce a substantial
fraction, typically at least 20 percent or more, of incompletely
extended amplification products. The amplification products,
including the incompletely extended amplification products are
denatured and subjected to at least one additional cycle of
reannealing and amplification. This variation, wherein at least one
cycle of reannealing and amplification provides a substantial
fraction of incompletely extended products, is termed "stuttering."
In the subsequent amplification round, the incompletely extended
products anneal to and prime extension on different
sequence-related template species.
[0166] In a further variation, at least one cycle of amplification
can be conducted using a collection of overlapping single-stranded
DNA fragments of related sequence, and different lengths. Each
fragment can hybridize to and prime polynucleotide chain extension
of a second fragment from the collection, thus forming
sequence-recombined polynucleotides. In a further variation,
single-stranded DNA fragments of variable length can be generated
from a single primer by Vent DNA polymerase on a first DNA
template. The single stranded DNA fragments are used as primers for
a second, Kunkel-type template, consisting of a uracil-containing
circular single-stranded DNA. This results in multiple
substitutions of the first template into the second (see Levichkin
et al., Mol. Biology 9:572-577 (1995)).
[0167] Nucleic acid sequences can be recombined by recursive
sequence recombination even if they lack sequence homology.
Homology can be introduced using synthetic oligonucleotides as PCR
primers. In addition to the specific sequences for the nucleic acid
segment being amplified, all of the primers used to amplify one
particular segment are synthesized to contain an additional
sequence of 20-40 bases 5' to the gene (sequence A) and a different
20-40 base sequence 3' to the segment (sequence B). An adjacent
segment is amplified using a 5' primer which contains the
complementary strand of sequence B (sequence B'), and a 3' primer
containing a different 20-40 base sequence (C). Similarly, primers
for the next adjacent segment contain sequences C' (complementary
to C) and D. In this way, small regions of homology are introduced,
making the segments into site-specific recombination cassettes.
Subsequent to the initial amplification of individual segments, the
amplified segments can then be mixed and subjected to primerless
PCR.
[0168] When domains within a polypeptide are shuffled, it may not
be possible to introduce additional flanking sequences to the
domains, due to the constraint of maintaining a continuous open
reading frame. Instead, groups of oligonucleotides are synthesized
that are homologous to the 3' end of the first domain encoded by
one of the genes to be shuffled, and the 5' ends of the second
domains encoded by all of the other genes to be shuffled together.
This is repeated with all domains, thus providing sequences that
allow recombination between protein domains while maintaining their
order.
[0169] B. In Vivo Formats
[0170] 1. Plasmid-Plasmid Recombination
[0171] The initial substrates for recombination are a collection of
polynucleotides comprising variant forms of a gene. The variant
forms usually/show substantial sequence identity to each other
sufficient to allow homologous recombination between substrates.
The diversity between the polynucleotides can be nature e.g.,
allelic or species variants), induced (e.g., error-prone PCR or
error-prone recursive sequence recombination), or the result of in
vitro recombination. Diversity can also result from resynthesizing
genes encoding natural proteins with alternative codon usage. There
should be at least sufficient diversity between substrates that
recombination can generate more diverse products than there are
starting materials. There must be at least two substrates differing
in at least two positions. However, commonly a library of
substrates of 10.sup.3-10.sup.8 members is employed. The degree of
diversity depends on the length of the substrate being recombined
and the extent of the functional change to be evolved. Diversity at
between 0.1-25% of positions is typical. The diverse substrates are
incorporated into plasmids. The plasmids are often standard cloning
vectors, e.g., bacterial multicopy plasmids. However, in some
methods to be described below, the plasmids include mobilization
(MOB) functions. The substrates can be incorporated into the same
or different plasmids. Often at least two different types of
plasmid having different types of selectable markers are used to
allow selection for cells containing at least two types of vector.
Also, where different types of plasmid are employed, the different
plasmids can come from two distinct incompatibility groups to allow
stable co-existence of two different plasmids within the cell.
Nevertheless, plasmids from the same incompatibility group can
still co-exist within the same cell for sufficient time to allow
homologous recombination to occur.
[0172] Plasmids containing diverse substrates are initially
introduced into cells by any method (e.g., chemical transformation,
natural competence, electroporation, biolistics, packaging into
phage or viral systems). Often, the plasmids are present at or near
saturating concentration (with respect to maximum transfection
capacity to increase the probability or more than one plasmid
entering the same cell. The plasmids containing the various
substrates can be transfected simultaneously or in multiple rounds.
For example, in the latter approach cells can be transfected with a
first aliquot of plasmid, transfectants selected and propagated,
and then infected with a second aliquot of plasmid.
[0173] Having introduced the plasmids into cells, recombination
between substrates to generate recombinant genes occurs within
cells containing multiple different plasmids merely by propagating
the cells. However, cells that receive only one plasmid are unable
to participate in recombination and the potential contribution of
substrates on such plasmids to evolution is not fully exploited
(although these plasmids may contribute to some extent if hey are
progagated in mutator cells). The rate of evolution can be
increased by allowing all substrates to participate in
recombination. Such can be achieved by subjecting transfected cells
to electroporation. The conditions for electroporation are the same
as those conventionally used for introducing exogenous DNA into
cells (e.g., 1,000-2,500 volts, 400 .mu.F and a 1-2 mM gap). Under
these conditions, plasmids are exchanged between cells allowing all
substrates to participate in recombination. In addition the
products of recombination can undergo further rounds of
recombination with each other or with the original substrate. The
rate of evolution can also be increased by use of conjugative
transfer. To exploit conjugative transfer, substrates can be cloned
into plasmids having MOB genes, and tra genes are also provided in
cis or in trans to the MOB genes. The effect of conjugative
transfer is very similar to electroporation in that it allows
plasmids to move between cells and allows recombination between any
substrate and the products of previous recombination to occur,
merely by propagating the culture. The rate of evolution can also
be increased by fusing cells to induce exchange of plasmids or
chromosomes. Fusion can be induced by chemical agents, such as PEG,
or viral proteins, such as influenza virus hemagglutinin, HSV-1 gB
and gD. The rate of evolution can also be increased by use mutator
host cells e.g., Mut L, S, D, T, H in bacteria and Ataxia
telangiectasia human cell lines).
[0174] The time for which cells are propagated and recombination is
allowed to occur, of course, varies with the cell type but is
generally not critical, because even a small degree of
recombination can substantially increase diversity relative to the
starting materials. Cells bearing plasmids containing recombined
genes are subject to screening or selection for a desired function.
For example, if the substrate being evolved contains a drug
resistance gene, one would select for drug resistance. Cells
surviving screening or selection can be subjected to one or more
rounds of screening/selection followed by recombination or can be
subjected directly to an additional round of recombination.
"Screening" as used herein is intended to include "selection" as a
type of screen.
[0175] The next round of recombination can be achieved by several
different formats independently of the previous round. For example,
a further round of recombination can be effected simply by resuming
the electroporation or conjugation-mediated intercellular transfer
of plasmids described above. Alternatively, a fresh substrate or
substrates, the same or different from previous substrates, can be
transfected into cells surviving selection/screening. Optionally
the new substrates are included in plasmid vectors bearing a
different selective marker and/or from a different incompatibility
group than the original plasmids. As a further alternative, cells
surviving selection/screening can be subdivided into two
subpopulations, and plasmid DNA from one subpopulation transfected
into the other, where the substrates from the plasmids from the two
subpopulations undergo a further round of recombination. In either
of the latter two options, the rate of evolution can be increased
by employing DNA extraction, electroporation, conjugation or
mutator cells, as described above. In a still further variation,
DNA from cells surviving screening/selection can be extracted and
subjected to in vitro recursive sequence recombination.
[0176] After The second round recombination, a second round of
screening/selection is performed, preferably under conditions of
increased stringency. If desired, further rounds of recombination
and selection/screening can be performed using the same strategy as
for the second round. With successive rounds of recombination and
selection/screening, the surviving recombined substrates evolve
toward acquisition of a desired phenotype. Typically, in this and
other methods of recursive recombination, the final product of
recombination that has acquired the desired phenotype differs from
starting substrates at 0.1%-25% of positions and has evolved at a
rate orders of magnitude in excess (e.g., by at least 10-fold,
100-fold, 1000-fold, or 10,000 fold) of the rate of evolution
driven by naturally acquired mutation of about 1 mutation per
10.sup.-9 positions per generation (see Anderson et al., Proc.
Natl. Acad. Sci. U.S.A. 93:906-907 (1996)). The "final product" may
be transferred to another host more desirable for utilization of
the "shuffled" DNA. This is particularly advantageous in situations
where the more desirable host is less efficient as a host for the
many cycles of mutation/recombination due to the lack of molecular
biology or genetic tools available for other organisms such as E.
coli.
[0177] 2. Virus-Plasmid Recombination
[0178] The strategy used for plasmid-plasmid recombination can also
be used for virus-plasmid recombination; usually, phage-plasmid
recombination. However, some additional comments particular to the
use of viruses are appropriate. The initial substrates for
recombination are cloned into both plasmid and viral vectors. It is
usually not critical which substrate(s) is/are inserted into the
viral vector and which into the plasmid, although usually the viral
vector should contain different substrate(s) from the plasmid. As
before, the plasmid (and the virus, typically contains a selective
marker. The plasmid and viral sectors can both be introduced into
cells by transfection as described above. However, a more efficient
procedure is to transfect the cells with plasmid, select
transfectants ant infect the transfectants with virus. Because the
efficiency of infection of many viruses approaches 100% of cells,
most cells transfected and infected by this route contain both a
plasmid and virus bearing different substrates.
[0179] Homologous recombination occurs between plasmid and virus
generating both recombined plasmids and recombined virus. For some
viruses, such as filamentous phage, in which intracellular DNA
exists in both double-stranded and single-stranded forms, both can
participate in recombination. Provided that the virus is not one
that rapidly kills cells, recombination can be augmented by use of
electroporation or conjugation to transfer plasmids between cells.
Recombination can also be augmented for some types of virus by
allowing the progeny virus from one cell to reinfect other cells.
For some types of virus, virus infected-cells show resistance to
superinfection. However, such resistance can be overcome by
infecting at high multiplicity and/or using mutant strains of the
virus in which resistance to superinfection is reduced.
[0180] The result of infecting plasmid-containing cells with virus
depends on the nature of the virus. Some viruses, such as
filamentous phage, stably exist with a plasmid in the cell and also
extrude progeny phage from the cell. Other viruses, such as lambda
having a cosmid genome, stably exist in a cell like plasmids
without producing progeny virions. Other viruses, such as the
T-phage and lytic lambda, undergo recombination with the plasmid
but ultimately kill the host cell and destroy plasmid DNA. For
viruses that infect cells without killing the host, cells
containing recombinant plasmids and virus can be screened/selected
using the same approach as for plasmid-plasmid recombination.
Progeny virus extruded by cells surviving selection/screening can
also be collected and used as substrates in subsequent rounds of
recombination. For viruses kill their host cells, recombinant genes
resulting from recombination reside only in the progeny virus. If
the screening or selective assay requires expression of recombinant
genes in a cell, the recombinant genes should be transferred from
the progeny virus to another vector, e.g., a plasmid vector, and
retransfected into cells before selection/screening is
performed.
[0181] For filamentous phage, the products of recombination are
present in both cells surviving recombination and in phage extruded
from these cells. The dual source of recombinant products provides
some additional options relative to the plasmid-plasmid
recombination. For example, DNA can be isolated from phage
particles for use in a round of in vitro recombination.
Alternatively, the progeny phage can be used to transfect or infect
cells surviving a previous round of screening/selection, or fresh
cells transfected with fresh substrates for recombination.
[0182] 3. Virus-Virus Recombination
[0183] The principles described for plasmid-plasmid and
plasmid-viral recombination can be applied to virus-virus
recombination with a few modifications. The initial substrates for
recombination are cloned into a viral vector. Usually, the same
vector is used for all substrates. Preferably, the virus is one
that, naturally or as a result of mutation, does not kill cells.
After insertion, some viral genomes can be packaged in vitro or
using a packaging cell line. The packaged viruses are used to
infect cells at high multiplicity such that there is a high
probability that a cell will receive multiple viruses bearing
different substrates.
[0184] After the initial round of infection, subsequent steps
depend on the nature of infection as discussed in the previous
section. For example, if the viruses have phagemid (Sambrook et
al., Molecular Cloning, CSH Press, 1987) genomes such as lambda
cosmids or M13, F1 or Fd phagemids, the phagemids behave as
plasmids within the cell and undergo recombination simply by
propagating the cells. Recombination is particularly efficient
between single-stranded forms of intracellular DNA. Recombination
can be augmented by electroporation of cells.
[0185] Following selection/screening, cosmids containing
recombinant genes can be recovered from surviving cells, e.g., by
heat induction of a cos.sup.-lysogenic host cell, or extraction of
DNA by standard procedures, followed by repackaging cosmid DNA in
vitro.
[0186] If the viruses are filamentous phage, recombination of
replicating form DNA occurs by propagating the culture of infected
cells. Selection/screening identifies colonies of cells containing
viral vectors having recombinant genes with improved properties,
together with infectious particles i.e., phage or packaged
phagemids) extruded from such cells. Subsequent options are
essentially the same as for plasmid-viral recombination.
[0187] 4. Chromosome Recombination
[0188] This format can be used to especially evolve chromosomal
substrates. The format is particularly preferred in situations in
which many chromosomal genes contribute to a phenotype or one does
not know the exact location of the chromosomal gene(s) to be
evolved. The initial substrates for recombination are cloned into a
plasmid vector. If the chromosomal gene(s) to be evolved are known,
the substrates constitute a family of sequences showing a high
degree of sequence identity but some divergence from the
chromosomal gene. If the chromosomal genes to be evolved have not
been located, the initial substrates usually constitute a library
of DNA segments of which only a small number show sequence identity
to the gene or gene(s) to be evolved. Divergence between
plasmid-borne substrate and the chromosomal gene(s) can be induced
by mutagenesis or by obtaining the plasmid-borne substrates from a
different species than that of the cells bearing the
chromosome.
[0189] The plasmids bearing substrates for recombination are
transfected into cells having chromosomal gene(s) to be evolved.
Evolution can occur simply by propagating the culture, and can be
accelerated by transferring plasmids between cells by conjugation
or electroporation. Evolution can be further accelerated by use of
mutator host cells or by seeding a culture of nonmutator host cells
being evolved with mutator host cells and inducing intercellular
transfer of plasmids by electroporation or conjugation. Preferably,
mutator host cells used for seeding contain a negative selectable
marker to facilitate isolation of a sure culture of the nonmutator
cells being evolved. Selection/screening identifies cells bearing
chromosomes and/or plasmids that have evolved toward acquisition of
a desired function.
[0190] Subsequent rounds of recombination and selection/screening
proceed in similar fashion to those described for plasmid-plasmid
recombination. For example, further recombination can be effected
by propagating cells surviving recombination in combination with
electroporation or conjugative transfer of plasmids. Alternatively,
plasmids bearing additional substrates for recombination can be
introduced into the surviving cells. Preferably, such plasmids are
from a different incompatibility group and bear a different
selective marker than the original plasmids to allow selection for
cells containing at least two different plasmids. As a further
alternative, plasmid and/or chromosomal DNA can be isolated from a
subpopulation of surviving cells and transfected into a second
subpopulation. Chromosomal DNA can be cloned into a plasmid vector
before transfection.
[0191] 5. Virus-Chromosome Recombination
[0192] As in the other methods described above, the virus is
usually one that does not kill the cells, and is often a phage or
phagemid. The procedure is substantially the same as for
plasmid-chromosome recombination. Substrates for recombination are
cloned into the vector. Vectors including the substrates can then
be transfected into cells or in vitro packaged and introduced into
cells by infection. Viral genomes recombine with host chromosomes
merely by propagating a culture. Evolution can be accelerated by
allowing intercellular transfer of viral genomes by
electroporation, or reinfection of cells by progeny virions.
Screening/selection identifies cells having chromosomes and/or
viral genomes that have evolved toward acquisition of a desired
function.
[0193] There are several options for subsequent rounds of
recombination. For example, viral genomes can be transferred
between cells surviving selection/recombination by electroporation.
Alternatively, viruses extruded from cells surviving
selection/screening can be pooled and used to superinfect the cells
at high multiplicity. Alternatively, fresh substrates for
recombination can be introduced into the cells, either on plasmid
or viral vectors.
[0194] II. Application of Recursive Sequence Recombination to
Evolution of Polypeptides
[0195] In addition to the techniques described above, some
additionally advantageous modifications of these techniques for the
evolution of polypeptides are described below. These methods are
referred to as "fine grain" and "coarse grain" shuffling. The
coarse grain methods allow one to exchange chunks of genetic
material between substrate nucleic acids, thereby limiting
diversity in the resulting recombinants to exchanges or
substitutions of domains, restriction fragments, oligo-encoded
blocks of mutations, or other arbitrarily defined segments, rather
than introducing diversity more randomly across the substrate. In
contrast to coarse grain shuffling, fine grain shuffling methods
allow the generation of all possible recombinations, or
permutations, of a given set of very closely linked mutations,
including multiple permutations, within a single segment, such as a
codon.
[0196] In some embodiments, coarse grain or fine grain shuffling
techniques are not performed as exhaustive searches of all possible
mutations within a nucleic acid sequence. Rather, these techniques
are utilized to provide a sampling of variation possible within a
gene based on known sequence or structural information. The size of
the sample is typically determined by the nature of the screen or
selection process. For example, when a screen is performed in a
95-well microtiter format, it may be preferable to limit the size
of the recombinant library to about 100 such microtiter plates for
convenience in screening.
[0197] A. Use of Restriction Enzyme Sites to Recombine
Mutations
[0198] In some situations it is advantageous to use restriction
enzyme sites in nucleic acids to direct the recombination of
mutations in a nucleic acid sequence of interest. These techniques
are particularly preferred in the evolution of fragments that
cannot readily be shuffled by existing methods due to the presence
of repeated DNA or other problematic primary sequence motifs. They
are also preferred for shuffling large fragments (typically greater
than 10 kb), such as gene clusters that cannot be readily shuffled
and "PCR-amplified" because of their size. Although fragments up to
50 kb have been reported to be amplified by PCR (Barnes, Proc.
Natl. Acad. Sci. (U.S.A.) 91:2216-2220 (1994)), it can be
problematic for fragments over 10 kb, and thus alternative methods
for shuffling in the range of 10 -50 kb and beyond are preferred.
Preferably, the restriction endonucleases used are of the Class II
type (Sambook et al., Molecular Cloning, CSH Press, 1987) and of
these, preferably those which generate nonpalindromic sticky end
overhangs such as Alwn I, Sfi I or BstX1. These enzymes generate
nonpalindromic ends that allow for efficient ordered reassembly
with DNA ligase. Typically, restriction enzyme (or endonuclease)
sites are identified by conventional restriction enzyme mapping
techniques (Sambrook et al., Molecular Cloning, CSH Press, 1987),
by analysis of sequence information for that gene, or by
introduction of desired restriction sites into a nucleic acid
sequence by synthesis (i.e. by incorporation of silent
mutations).
[0199] The DNA substrate molecules to be digested can either be
from in vivo replicated DNA, such as a plasmid preparation, or from
PCR amplified nucleic acid fragments harboring the restriction
enzyme recognition sites of interest, preferable near the ends of
the fragment. Typically, at least two variants of a gene of
interest, each having one or more mutations, are digested with at
least one restriction enzyme determined to cut within the nucleic
acid sequence of interest. The restriction fragments are then
joined with DNA ligase to generate cull length genes having
shuffled regions. The number of regions shuffled will depend on the
number of cuts within the nucleic acid sequence of interest. The
shuffled molecules can be introduced into cells as described above
and screened or selected for a desired property. Nucleic acid can
then be isolated from pools (libraries) or clones having desired
properties and subjected to the same procedure until a desired
degree of improvement is obtained.
[0200] In some embodiments, at least one DNA substrate molecule or
fragment thereof is isolated and subjected to mutagenesis. In some
embodiments, the pool or library of religated restriction fragments
are subjected to mutagenesis before the digestion-ligation process
is repeated. "Mutagenesis" as used herein comprises such techniques
known in the art as PCR mutagenesis, oligonucleotide-directed
mutagenesis, site-directed mutagenesis, etc., and recursive
sequence recombination by any of the techniques described
herein.
[0201] An example of the use of this format is in the manipulation
of polyketide clusters. Polyketide clusters (Khosla et al., TIBTECH
14, September 1996) are typically 10 to 100 kb in length,
specifying multiple large polypeptides which assemble into very
large multienzyme complexes. Due to the modular nature of these
complexes and the modular nature of the biosynthetic pathway,
nucleic acids encoding protein modules can be exchanged between
different polyketide clusters to generate novel and functional
chimeri, polyketides. The introduction of rare restriction
endonuclease sites such as SfiI (eight base recognition,
nonpalindromic overhangs) at nonessential sites between
polypeptides or in introns engineered within polypeptides would
provide "handles" with which to manipulate exchange of nucleic acid
segments using the technique described above.
[0202] B. Reassembly PCR
[0203] A further technique for recursively recombining mutations in
a nucleic acid sequence utilizes "reassembly PCR". This method can
be used to assemble multiple segments that have been separately
evolved into a full length nucleic acid template such as a gene.
This technique is performed when a pool of advantageous mutants is
known from previous work or has been identified by screening
mutants that may have been created by any mutagenesis technique
known in the art, such as PCR mutagenesis, cassette mutagenesis,
doped oligo mutagenesis, chemical mutagenesis, or propagation of
the DNA template in vivo in mutator strains. Boundaries defining
segments of a nucleic acid sequence of interest preferably lie in
intergenic regions, introns, or areas of a gene not likely to have
mutations of interest. Preferably, oligonucleotide primers (oligos)
are synthesized for PCR amplification of segments of the nucleic
acid sequence of interest, such that the sequences of the
oligonucleotides overlap the junctions of two segments. The overlap
region is typically about 10 to 100 nucleotides in length. Each of
the segments is amplified with a set of such primers. The PCR
products are then "reassembled" according to assembly protocols
such as those used in Sections IA-B above to assemble randomly
fragmented genes. In brief, in an assembly protocol the PCR
products are first purified away from the primers, by, for example,
gel electrophoresis or size exclusion chromatography. Purified
products are mixed together and subjected to about 1-10 cycles of
denaturing, reannealing, and extension in the presence of
polymerase and deoxynucleoside triphosphates (dNTP's) and
appropriate buffer salts in the absence of additional primers
("self-priming" ). Subsequent PCR with primers flanking the gene
are used to amplify the yield of the fully reassembled and shuffled
genes. This method is necessarily "coarse grain" and hence only
recombines mutations in a clockwise fashion, an advantage for some
searches such as when recombining allelic variants of multiple
genes within an operon.
[0204] In some embodiments, the resulting reassembled genes are
subjected to mutagenesis before the process is repeated.
[0205] In some embodiments, oligonucleotides that incorporate
uracil into the primers are used for PCR amplification. Typically
uracil is incorporated at one site in the oligonucleotide. The
products are created with uracil glycosylase, thereby generating a
single-stranded overhang, and are reassembled in an ordered fashion
by a method such as disclosed by Rashtchian (Current Biology
5:30-36 (1995)).
[0206] In a further embodiment, the PCR primers for amplification
of segments of the nucleic acid sequence of interest are used to
introduce variation into the gene of interest as follows. Mutations
at sites of interest in a nucleic acid sequence are identified by
screening or selection, by sequencing homologues of the nucleic
acid sequence, and so on. Oligonucleotide PCR primers are then
synthesized which encode wild type or mutant information at sites
of interest. These primers are then used in PCR mutagenesis to
generate libraries of full length genes encoding permutations of
wild type and mutant information at the designated positions. This
technique is typically advantagous in cases where the screening or
selection process is expensive, cumbersome, or impractical relative
to the cost of sequencing the genes of mutants of interest and
synthesizing mutagenic oligonucleotides.
[0207] An example of this method is the evolution of an improved
Taq polymerase, as described in detail below. Mutant proteins
resulting from application of the method are identified and assayed
in a sequencing reaction to identify mutants with improved
sequencing properties. This is typically done in a high throughput
format (see, for example, Broach et al. Nature 384 (Supp): 14-16
(1996)) to yield, after screening, a small number, e.g., about 2 to
100, of candidate recombinants for further evaluation. The mutant
genes can then be sequenced to provide information regarding the
location of the mutation. The corresponding mutagenic
oligonucleotide primers can be synthesized from this information,
and used in a reassembly reaction as described above to efficiently
generate a library with an average of many mutations per gene.
Thus, multiple rounds of this protocol allows the efficient search
for improved variants of the Taq polymerase.
[0208] C. Enrichment for Mutant Sequence Information
[0209] In some embodiments of the invention, recombination
reactions, such as those discussed above, are enriched for mutant
sequences so that the multiple mutant spectrum, i.e. possible
combinations of mutations, is more efficiently sampled. The
rationale for this is as follows. Assume that a number, n, of
mutant clones with improved activity is obtained, wherein each
clone has a single point mutation at a different position in the
nucleic acid sequence. If this population of mutant clones with an
average of one mutation of interest per nucleic acid sequence is
then put into a recombination reaction, the resulting population
will still have an average of one mutation of interest per nucleic
acid sequence as defined by a Poisson distribution, leaving the
multiple mutation spectrum relatively unpopulated.
[0210] The amount of screening required to identify recombinants
having two or more mutations can be dramatically reduced by the
following technique. The nucleic acid sequences of interest are
obtained from a pool of mutant clones and prepared as fragments,
typically by digestion with a restriction endonuclease, sonication,
or by PCR amplification. The fragments are denatured, then allowed
to reanneal, thereby generating mismatched hybrids where one strand
of a mutant has hybridized with a complementary strand from a
different mutant or wild-type clone. The reannealed products are
then fragmented into fragments of about 20 -100 bp, for example, by
the use of DNAseI. This fragmentation reaction has the effect of
segregation regions of the template containing mismatches mutant
information from those encoding wild type sequence. The mismatched
hybrids can then be affinity purified using aptamers, dyes, or
other agents which bind to mismatched DNA. A preferred embodiment
is the use of mutS protein affinity matrix Wagner et al., Nucleic
Acids Res. 23(19):3944-3948 (1995); Su et al., Proc. Natl. Acad.
Sci. (U.S.A.), 83:5057-5061(1986)) with a preferred step of
amplifying the affinity-purified maternal in vitro prior to an
assembly reaction. This amplified material is then put into a
assembly PCR reaction as decribed above. Optionally, this material
can be titrated against the original mutant pool (e.g., from about
100 to 10% of the mutS enriched pool) to control the average number
of mutations per clone in the next round of recombination.
[0211] Another application of this method is in the assembly of
gene constructs that are enriched for polymorphic bases occurring
as natural or selected allelic variants or as differences between
homologous genes of related species. For example, one may have
several varieties of a plant that are believed to have heritable
variation in a trait of interest (e.g., drought resistance). It
then is of interest to construct a library of these variant genes
containing many mutations per gene. MutS selection can be applied
in combination with the assembly techniques described herein to
generate such a pool of recombinants that are highly enriched for
polymorphic ("mutant") information. In some embodiments, the pool
of recombinant genes is provided in a transgenic host. Recombinants
can be further evolved by PCR amplification of the transgene from
transgenic organisms that are determined to have an improved
phenotype and applying the formats described in this invention to
further evolve them.
[0212] D. Intron-driven Recombination
[0213] In some instances, the substrate molecules for recombination
have uniformly low homology, sporadically distributed regions of
homology, or the region of homology s relatively small for example,
about 10-100 bp), such as phage displayed peptide ligants. These
factors can reduce the efficiency and randomness of recombination
in RSR. In some embodiments of the invention, this problem is
addressed by the introduction of introns between coding exons in
sequences encoding protein homologues. In embodiments of the
invention, introns can be used (Chong et al., J. Biod. Chem.,
271:22159-22168 (1996)).
[0214] In this method, a nucleic acid sequence, such as a gene or
gene family, is arbitrarily defined to have segments. The segments
are preferably exons. Introns are engineered between the segments.
Preferably, the intron inserted between the first and second
segments is at least about 10% divergent from the intron inserted
between second and third segments, the intron inserted between
second and third segments is at least about 10% divergent from the
introns inserted between any of the previous segment pairs, and so
on through segments n and n+1. The introns between any given set of
exons will thus initially be identical between all clones in the
library, whereas the exons can be arbitrarily divergent in
sequence. The introns therefore provide homologous DNA sequences
that will permit application of any of the described methods for
RSR while the exons can be arbitrarily small or divergent in
sequence, and can evolve to achieve an arbitrarily large degree of
sequence divergence without a significant loss in efficiency in
recombination. Restriction sites can also be engineered into the
intronic nucleic acid sequence of interest so as to allow a
directed reassemmbly of restriction fragments. The starting exon
DNA may be synthesized de novo from sequence information, or may be
present in any nucleic acid preparation ( e.g., genomic, cDNA,
libraries, and so on). For example, 1 to 10 nonhomologous introns
can be designed to direct recombination of the nucleic acid
sequences of interest by placing them between exons. The sequence
of the introns can be all or partly obtained from known intron
sequence. Preferably, the introns are self-splicing. Ordered sets
of introns and exon libraries are assembled into functional genes
by standard methods (Sambrook et al., Molecular Cloning, CSH Press
(1987)).
[0215] Any of the formats for in vitro or in vivo recombination
described herein can be applied for recursive exon shuffling. A
preferred format is to use nonpalindromic restriction sites such as
Sfi I placed into the intronic sequences to promote shuffling.
Pools of selected clones are digested with Sfi I and religated. The
nonpalindromic overhangs promote ordered reassembly of the shuffled
exons. These libraries of genes can be expressed and screened for
desired properties, then subjected to further recursive rounds of
recombination by this process. In some embodiments, the libraries
are subjected to mutagenesis before the process is repeated.
[0216] An example of how the introduction of an intron into a
mammalian library format would be used advantageously is as
follows. An intron containing a lox (Sauer et al., Proc. Natl.
Acad. Sci. (U.S.A.), 85:5166-5170 (1988)) site is arbitrarily
introduced between amino acids 92 and 93 in each alpha interferon
parental substrate. A library of 10.sup.4 chimeric interferon genes
is made for each of the two exons (residues 1-92 and residues
93-167), cloned into a replicating plasmid vector, and introduced
into target cells. The number 10.sup.4 is arbitrarily chosen for
convenience in screening. An exemplary vector for expression in
mammalian cells would contain an SV40 origin, with the host cells
expressing SV40 large T antigen, so as to allow transient
expression of the interferon constructs. The cells are challenged
with a cytopathic virus such as vesicular stomatitis virus (VSV) in
an interferon protection assay (e.g., Meister et al., J. Gen.
Virol. 67:1633-1643, (1986)). Cells surviving due to expression of
interferon are recovered, the two libraries of interferon genes are
PCR amplified, and recloned into a vector that can be amplified in
E. coli. The amplified plasmids are then transfected at high
multiplicity (e.g. 10 micrograms of plasmid per 10.sup.6 cells)
into a cre expressing host that can support replication of that
vector. The presence of cre in the host cells promotes efficient
recombination at the lox site in the interferon intron, thus
shuffling the selected sets of exons. This population of cells is
then used in a second round of selection by viral challenge and the
process is applied recursively. In this format, the cre recombinase
is preferrably expressed transiently on a cotransfected molecule
that cannot replicate in the host. Thus, after segregation of
recombinants from the cre expressing plasmid, no further
recombination will occur and selection can be performed on
genetically stable exon permutations. The method can be used with
more than one intron, with recombination enhancing sequences other
than cre/lox (e.g., int/xis, etc.), and with other vector systems
such as but not limited to retroviruses, adenovirus or
adeno-associated virus.
[0217] 5. Synthetic Oligonucleotide Mediated Recombination
[0218] 1. Oligo bridge across sequence space
[0219] In some embodiments of the invention, a search of a region
of sequence space defined by a set of substrates, such as members
of a gene family, having less than about 80%, more typically, less
than about 50% homology, is desired. This region, which can be part
or all of a gene or a gene is arbitrarily delineated into segments.
The segment borders can be chosen randomly, based on correspondence
with natural exons, based on structural considerations (loops,
alpha helices, subdomains, whole domains, hydrophobic core,
surface, dynamic simulations), and based on correlations with
genetic mapping data.
[0220] Typically, the segments are then amplified by PCR with a
pool of "bridge" oligonucleotides at each junction. Thus, if the
set of five genes is broken into three segments A, B and C, and if
there are five versions of each segment (A1, A2, . . . C4, C5),
twenty five oligonucleotides are made for each strand of the A-B
junctions where each bridge oligo has 20 bases of homology to one
of the A and one of the B segments. In some cases, the number of
required oligonucleotides can be reduced by choosing segment
boundaries that are identical in some or all of the gene family
members. Oligonucleotides are similarly synthesized for the B-C
junction. The family of A domains is amplified by PCR with an
outside generic A primer and the pool of A-B junction
oligonucleotides; the B domains with the A-B plus the B-C bridge
oligonucleotides, and the C domains with the B-C bridge
oligonucleotides plus a generic outside primer. Full length genes
are made then made by assembly PCR or by the dUTP/uracil
glycosylase methods described above. Preferably, products from this
step are subjected to mutagenesis before the process of selection
and recombination is repeated, until a desired level of improvement
or the evolution of a desired property is obtained. This is
typically determined using a screening or selection as appropriate
for the protein and property of interest.
[0221] An illustration of this method is illustrated below for the
recombination of eleven homologous human alpha interferon
genes.
[0222] 2. Site Directed Mutagenesis (SDM) with Oligonucleotides
Encoding Homologue Mutations Followed by Shuffling
[0223] In some embodiments of the invention, sequence information
from one or more substrate sequences is added to a given "parental"
sequence of interest, with subsequent recombination between rounds
of screening or selection. Typically, this is done with
site-directed mutagenesis performed by techniques well known in the
art (Sambrook et al., Molecular Cloning, CSH Press (1987)) with one
substrate as template and oligonucleotides encoding single or
multiple mutations from other substrate sequences, e.g. homologous
genes. After screening or selection for an improved phenotype of
interest, the selected recombinant(s) can be further evolved using
RSR techniques described herein. After screening or selection,
site-directed mu agenesis can be done again with another collection
of oligonucleotides encoding homologue mutations, and the above
process repeated until the desired properties are obtained.
[0224] When the difference between two homologues is one or more
single point mutations in a codon, degenerate oligonucleotides can
be used that encode the sequences in both homologues. One oligo may
include many such degenerate codons and still allow one to
exhaustively search all permutations over that block of sequence.
An example of this is provided below for the evolution of alpha
interferon genes.
[0225] When the homologue sequence space is very large, it can be
advantageous to restrict the search to certain variants. Thus, for
example, computer modelling tools (Lathrop et al., J. Mol. Biol.,
255:641-665 (1996)) can be used to model each homologue mutation
onto the target protein and discard any mutations that are
predicted to grossly disrupt structure and function.
[0226] F. Recombination Directed by Host Machinery
[0227] In some embodiments of the invention, DNA substrate
molecules are introduced into cells, wherein the cellular machinery
directs their recombination. For example, a library of mutants is
constructed and screened or selected for mutants with improved
phenotypes by any of the techniques described herein. The DNA
substrate molecules encoding the best candidates are recovered by
any of the techniques described herein, then fragmented and used to
transfect a mammalian host and screened or selected for improved
function. The DNA substrate molecules are recovered from the
mammalian host, such as by PCR, and the process is repeated until a
desired level of improvement is obtained. In some embodiments, the
fragments are denatured and reannealed prior to transfection,
coated with recombination stimulating proteins such as recA, or
co-transfected with a selectable marker such as NeoR to allow the
positive selection for cells receiving recombined versions of the
gene of interest.
[0228] For example, this format is preferred for the in vivo
affinity maturation of an antibody by RSR. In brief, a library of
mutant antibodies is generated, as described herein for the 48G7
affinity maturation. This library is FACS purified with ligand to
enrich for antibodies with the highest 0.1-10% affinity. The V
regions genes are recovered by PCR, fragmented, and cotransfected
or electorporated with a vector into which reassembled V region
genes can recombine. DNA substrate molecules are recovered from the
cotranfected cells, and the process is repeated until the desired
level of improvment is obtained. Other embodiments include
reassembling the V regions prior to the electroporation so that an
intact V region exon can recombine into an antibody expression
cassette. Further embodiments include the use of this format for
other eukaryotic genes or for the evolution of whole viruses.
[0229] G. Phagemid-Based Assembly
[0230] In some embodiments of the invention, a gene of interest is
cloned into a vector that generates single stranded DNA, such as a
phagemid. The resulting DNA substrate is mutagenzied by RSR in any
method known in the art, transfected into host cells, and subjected
to a screen or selection for a desired property or improved
phenotype. DNA from the selected or screened phagemids is
amplified, by, for example, PCR or plasmid preparation. This DNA
preparation contains the various mutant sequences that one wishes
to permute. This DNA is fragmented and denatured, and annealed with
single-stranded DNA (ssDNA) phagemid template (ssDNA encoding the
wild-type gene and vector sequences). A preferred embodiment is the
use of dut(-) ung(-) host strains such as CJ236 (Sambrook et al.,
Molecular Cloning CSH Press (1987)) for the preparation of
ssDNA.
[0231] Gaps in annealed template are filled with DNA polymerase and
ligated to form closed relaxed circles. Since multiple fragments
can anneal to the phagemid, the newly synthesized strand now
consists of shuffled sequences. These products are transformed into
a mutS strain of E. coli which is dut+ung+. Phagemid DNA is
recovered from the transfected host and subjected again to this
protocol until the desired level of improvement is obtained. The
gene encoding the protein of interest in this library of recovered
phagemid DNA can be mutagenzied by any technique, including RSR,
before the process is repeated.
[0232] II Improved Protein Expression
[0233] While recombinant DNA technology has proved to be a very
general method for obtaining large, pure, and homogeneous
quantities of almost all nucleic acid sequences of interest,
similar generality has not yet been achieved for the production
large amounts of pure, homogeneous protein in recombinant form,
likely explanation is that protein expression, folding,
localization and stability is intrinsically more complex and
unpredictable than for DNA. The yield of expressed protein is a
complex function of transcription rates, translation rates,
interactions with the ribosome, interaction of the nascent
polypeptide with chaperonins and other proteins in the cell,
efficiency of oligomerization, interaction with components of
secretion and other protein trafficking pathways, protease
sensitivity, and the intrinsic stability of the final folded state.
Optimization of such complex processes is well suited for the
application of RSR. The following methods detail strategies for
application of RSR to the optimization of protein expression.
[0234] A. Evolution of Mutant Genes with Improved Expression Using
RSR on Codon Usage Libraries
[0235] The negative effect of rare E. coli codons on expression of
recombinant proteins in this host has been clearly demonstrated
(Rosenberg, et al., J. Bact. 175:716-722 (1993)). However, general
rules for the choice of codon usage patterns to optimize expression
of functional protein have been elusive. In some embodiments of the
invention, protein expression is optimized by charging codons used
in the gene of interest, based on the degeneracy of the genetic
code. Typically, this is accomplished by synthesizing the gene
using degenerate oligonucleotides. In some embodiments the
degenerate oligonucleotides have the general structure of about 20
nucleotides of identity to a DNA substrate molecule encoding a
protein of interest, followed by a region of about 20 degenerate
nucleotides which encode a region of the protein, followed by
another region of about 20 nucleotides of identity. In a preferred
embodiment, the region of identity utilizes preferred codons or the
host. In a further embodiment, the oligonucleotides are identical
to the DNA substrate at least one 5' and one 3' nucleotide, but
have at least 85% sequence homology to the DNA substrate molecule,
with the difference due to the use of degenerate codons. In some
embodiments, a set of such degenerate oligonucleotides is used in
which each oligonucleotide overlaps with another by the general
formula n-10, wherein n the length of the oligonucleotide. Such
oligonucleotides are typically about 20-1000 nucleotides in length.
The assembled genes are then cloned, expressed, and screened or
selected for improved expression. The assembled genes can be
subjected to recursive recombination methods as descibed above
until the desired improvement is achieved.
[0236] For example, this technique can be used to evolve bovine
intestinal alkaline phosphatase (BIAP) for active expression in E.
coli. This enzyme is commonly used as a reporter gene in assay
formats such as ELISA. The cloned gene cannot be expressed in
active form in a prokaryotic host such as E. coli in good yield.
Development of such an expression system would allow one to access
inexpensive expression technology for BIAP and, importantly, for
engineered variants with improved activity or chemical coupling
properties (such as chemical coupling to antibodies) . A detailed
example is provide in the Experimental Examples section.
[0237] B. Improved Folding
[0238] In some embodiments of the invention, proteins of interest
when overexpressed or expressed in heterologous hosts form
inclusion bodies, with the majority of the expressed protein being
found in insoluble aggregates. Recursive sequence recombination
techniques can be used to optimize folding of such target proteins.
There are several ways to improve folding, including mutating
evolving the target protein of interest and evolving chaperonin
proteins.
[0239] 1. Evolving A Target Protein
[0240] a. Inclusion Body Fractionation Selection Using lac
Headpiece Dimer Fusion Protein
[0241] The lac repressor "headpiece dimer" is a small protein
containing two headpiece domains connected by a short peptide
linker which binds the lac operator with sufficient affinity that
polypeptide fusions to this headpiece dimer will remain bound the
plasmid that encodes them throughout an affinity purification
process (Gates et al., J. Mol. Biol. 255:373-386 (1995)). This
property can be exploited, as follows, to evolve mutant proteins of
interest with improved folding properties. The protein of interest
can be mammalian, yeast, bacterial, etc.
[0242] A fusion protein between the lac headpiece dimer and a
target protein sequence is constructed, for example, as disclosed
by Gates (supra). This construct, containing at least one lac
operator, is mutagenized by technologies common in the arts such as
PCR mutagenesis, chemical mutagenesis, oligo directed mutagenesis
(Sambrook et al., Molecular Cloning CSH Press (1987)). The
resulting library is transformed into a host cell, and expression
of the fusion protein is induced, preferably with arabinose. An
extract or lysate is generated from a culture of the library
expressing the construct. Insoluble protein is fractionated from
soluble protein/DNA complexes by centrifugation or affinity
chromatography, and the yield of soluble protein/DNA complexes is
quantitated by quantitative PCR (Sambrook et al., Molecular
Cloning, CSH Press, 1987) of the plasmid. Preferably, a reagent
that is specific for properly folded protein, such as a monoclonal
antibody or a natural ligand, is used to purify soluble protein/DNA
complexes. The plasmid DNA from this step is isolated, subjected to
RSR and again expressed. These steps are repeated until the yield
of soluble protein/DNA complexes has reached a desired level of
improvement. Individual clones are then screened for retention of
functional properties of the protein of interest, such as enzymatic
activity, etc.
[0243] This technique is generically useful for evolving solubility
and other properties such as cellular trafficking of proteins
heterologously expressed in a host cell of interest. For example,
one could select for efficient folding and nuclear localization of
a protein fused to the lac repressor headpiece dimer by encoding
the protein on a plasmid encoding an SV40 origin of replication and
a lac operator, and transiently expressing the fusion protein in a
mammalian host expressing T antigen. Purification of protein/DNA
complexes from nuclear HIRT extracts (Seed and Aruffo, Proc. Natl.
Acad. Sci. (U.S.A.), 34:3365-3369 (1987)) would allow one to select
o efficient folding and nuclear localization proteins.
[0244] b. Functional Expression of Protein Using Phage Display
[0245] A problem often encountered n phage display methods such as
those disclosed by O'Neil et al. (Current Biology, 5:443-449
(1995)) is the inability to functionally express a protein interest
on phage. Without being limited to any one theory, improper folding
of the protein of interest can be responsible for this problem. RSR
can be used to evolve a protein of interest for functional
expression on phage. Typically, a fusion protein is constructed
between gene III or gene VIII and the target protein and then
mutagenized, for example by PCR mutagenesis. The mutagenzied
library is then expressed in a phage display format, a phage lysate
is made, and these phage are affinity selected for those bearing
functionally displayed fusion proteins using an affinity matrix
containing a known ligand for the target protein.
[0246] DNA from the functionally selected phage is purified, and
the displayed genes of interest are shuffled and recloned into the
phage display format. The selection, shuffling and recloning steps
are repeated until the yield of phage with functional displayed
protein has reached desired levels as defined, for example, by the
fraction of phage that are retained on a ligand affinity matrix or
the biological activity associated with the displayed phage.
Individual clones are then screened to identify candidate mutants
with improved display properties, desired level of expression, and
functional properties of interest (e.g., ability to bind a ligand
or receptor, lymphokine activity, enzymatic activity, etc.).
[0247] In some embodiments of the invention, a functional screen or
selection is used to identify an evolved protein not expressed on a
phage. The target protein, which cannot initially be efficiently
expressed in a host of interest, is mutagenized and a functional
screen or selection is used to identify cells expressing functional
protein. For example, the protein of interest may complement a
function in the host cell, cleave a colorimetric substrate, etc.
Recursive sequence recombination is then used to rapidly evolve
improved functional expression from such a pool of improved
mutants.
[0248] For example, AMV reverse transcriptase is of particular
commercial importance because it is active at a higher temperature
(42.degree. C.) and is more robust than many other reverse
transcriptases. However, it is difficult to express in prokaryotic
hosts such as E. coli, and is consequently expensive because it has
to be purified from chicken cells. Thus an evolved AMV reverse
transcriptase that can be expressed efficiently in E. coli is
highly desirable.
[0249] In brief, the AMV reverse transcriptase gene (Papas et al.,
J. Cellular Biochem 20:95-103 (1982)) is mutagenized by any method
common in the art. The library of mutant genes is cloned into a
colE1 plasmid (Amp resistant) under control of the lac promoter in
a polA12 (Ts) recA718 (Sweasy et al. Proc. Natl. Acad. Sci. U.S.A.
90:4626-4630 (1993)) E. coli host. The library is induced with
IPTG, and shifted to the nonpermissive temperature. This selects
for functionally expressed reverse transcriptase genes under the
selective conditions reported for selection of active HIV reverse
transcriptase mutants reported by Kim et al. (Proc. Natl. Acad.
Sci. (U.S.A.), 92:684-688 (1995)). The selected AMV RTX genes are
recovered by PCR by using oligonucleotides flanking the cloned
gene. The resulting PCR products are subjected to in vitro RSR,
selected as described above, and the process is repeated until the
level of functional expression is acceptable. Individual clones are
then screened o RNA-dependent DNA polymerization and other
properties of interest (e.g. half life at room temperature, error
rate). The candidate clones are subjected to mutagenesis, and then
tested again to yield an AMV RT that can be expressed in E. coli at
high levels.
[0250] 2. Evolved Chaperonins
[0251] In some embodiments of the invention, overexpression of a
protein can lead to the accumulation of folding intermediates which
have a tendency to aggregate. Without being limited to any one
theory, the role of chaperonins is thought to be to stabilize such
folding intermediates against aggregration; thus, overexpression of
a protein of interest can lead to overwhelming the capacity of
chaperonins. Chaperonin genes can be evolved using the techniques
of the invention, either alone or in combination with the genes
encoding the protein of interest, to overcome this problem.
[0252] Examples of proteins of interest which are especially suited
to this approach include but are not limited to: cytokines;
malarial coat proteins; T cell receptors; antibodies; industrial
enzymes (e.g., detergent proteases and detergent lipases); viral
proteins for use in vaccines; and plant seed storage proteins.
[0253] Sources of chaperonin genes include but are limited to E.
coli chaperonin genes encoding such proteins as thioredoxin, Gro
ES/Gro EL, PapD, ClpB, DsbA, DsbB, DnaJ, DnaK, and GrpE; mammalian
chaperonins such as Hsp70, Hsp72, Hsp73, Hsp40, Hsp60, Hsp10, Hdj1,
TCP-1, Cpn60, BiP; and the homologues of these chaperonin genes in
other species such as yeast (J. G. Wall and A. Pluckthun, Current
Biology, 5:507-516 (1995); Hartl, Nature, 381:571-580 (1996)).
Additionally, heterologous genomic or cDNA libraries can be used as
libraries to select or screen for novel chaperonins.
[0254] In general, evolution of chaperonins is accomplished by
first mutagenizing chaperonin genes, screening or selecting for
improved expression of the target protein of interest, subjecting
the mutated chaperonin genes to RSR, and repeating selection or
screening. As with all RSR techniques, this is repeated until the
desired improvement of expression of the protein of interest is
obtained. Two exemplary approaches are provide below.
[0255] a. Chaperonin Evolution in Trans to the Protein of Interest
With a Screen or Selection for Improved Function
[0256] In some embodiments the chaperonin genes are evolved
independently of the gene s, for the protein of interest. The
improvement in the evolved chaperonin can be assayed, for example
by screening for enhancement of the activity of the target protein
itself or for the activity of a fusion protein comprising the
target protein and a selectable or screenable protein (e.g., GFP,
alkaline phosphatase or beta-galactosidase).
[0257] b. Chaperonin Operon in cis
[0258] In some embodiments, the chaperonin genes and the target
protein genes are encoded on the same plasmid, but not necessarily
evolved together. For example, a lac headpiece dimer can be fused
to the protein target to allow for selection of plasmids which
encode soluble protein. Chaperonin genes are provided on this same
plasmid ("cis") and are shuffled and evolved rather than the target
protein. Similarly, the chaperonin genes can be cloned onto a
phagemid plasmid that encodes a gene III or gene VIII fusion with a
protein of interest. The cloned chaperonins are mutagenized and, as
with the selection described above, phage expressing functionally
displayed fusion protein are isolated on an affinity matrix. The
chaperonin genes from these phage are shuffled and the cycle of
selection, mutation and recombination are applied recursively until
fusion proteins are efficiently displayed in functional form.
[0259] 3. Improved Intracellular Localization
[0260] Many overexpressed proteins of biotechnological interest are
secreted into the periplasm or media to give advantages in
purification or activity assays. Optimization for high level
secretion is difficult because the process is controlled by many
genes and hence optimization may require multiple mutations
affecting the expression level and structure of several of these
components. Protein secretion in E. coli, for example, is known to
be influenced by many proteins including: a secretory ATPase SecA),
a translocase complex SecB, SecD, SecE, SecF , and SecY;,
chaperonins (DnaK, DnaJ, GroES, GroEL. signal peptidases (LepB,
LspA, Ppp), specific folding catalysts DsbA and other proteins of
less well defined function e.g., Ffh, FtsY, Sandkvist et al. Curr.
Op. Biotechnol. 7:505-511 1996). Overproduction of wild type or
mutant copies of these genes for these proteins can significantly
increase the yield of mature secreted protein. For example,
overexpression of secY or secY4 significantly increased the
periplasmic yield of mature human IL6 from a hIL6-pre-OmpA fusion
(Perez-Perez et al., Bio-Technology 12:178-180 (1994)).
Analogously, overexpression of DnaK/DnaJ in E. coli improved the
yield of secreted human granulocyte colony stimulating factor
(Perez-Perez et al., Biochem. Biophys. Res. Commun. 210:254-259
(1995))
[0261] RSR provides a route to evolution of one or more of the
above named components of the secretory pathway. The following
strategy is employed to optimize protein secretion in E. coli.
Variations on this method, suitable for application to Bacillus
subtilis, Pseudomonas, Saccaromyces cerevisiae, Pichia pastoris,
mammalian cells and other hosts are also described. The general
protocol is as follows.
[0262] One or more of the genes named above are obtained by PCR
amplification from E. coli genomic DNA using known flanking
sequence, and cloned in an ordered array into a plasmid or cosmid
vector. These genes do not in general occur naturally in clusters,
and hence these will comprise artificial gene clusters. The genes
may be cloned under the control of their natural promoter or under
the control of another promoter such as the lac, tac, arabinose, or
trp promoters. Typically, rare restriction sites such as Sfi I are
placed between the genes to facilitate ordered reassembly of
shuffled genes as described in the methods of the invention.
[0263] The gene cluster is mutagenized and introduced into a host
cell in which the gene of interest can be inducibly expressed.
Expression of the target gene to be secreted and of the cloned
genes is induced by standard methods for the promoter of interest
(e.g., addition of 1 mM IPTG for the lac promoter). The efficiency
or protein secretion by a library of mutants is measured, for
example by the method of colony blotting (Skerra et al., Anal.
Biochem. 196:151-155 (1991). Those colonies expressing the highest
levels of secreted protein the top 0.1-10%; preferably the top 1%)
are picked. Plasmid DNA is prepared from these colonies and
shuffled according to any of the methods of the invention.
[0264] Preferably, each individual gene is amplified from the
population and subjected to RSR. The fragments are digested with
Sfi I (introduced between each gene with nonpalindromic overhangs
designed to promote ordered reassembly by DNA ligase) and ligated
together, preferably at low dilution to promote formation of
covalently closed relaxed circles (<1 ng/microliter). Each of
the PCR amplified gene populations may be shuffled prior to
reassembly into the final gene cluster. The ligation products are
transformed back into the host of interest and the cycle of
selection and RSR is repeated.
[0265] Analogous strategies can be employed in other hosts such as
Pseudomonas, Bacillus subtilis, yeast and mammalian cells. The
homologs of the E. coli genes listed above are targets for
optimization, and indeed many of these homologs have been
identified in other species (Pugsley, Microb. Rev. 57:50-108
(1993)). In addition to these homologs, other components such as
the six polypeptides of the signal recognition particle, the
trans-locating chain-associating membrane protein (TRAM), BiP, the
Ssa proteins and other hsp70 homologs, and prsA (B. subtilis)
(Simonen and Pulva, Microb. Rev. 57:109-137 (1993)) are targets for
optimization by RSR. In general, replicating episomal vectors such
as SV40-neo (Sambrook et al., Molecular Cloning, CSH Press (1987),
Northrup et al., J. Biol. Chem. 268(4):2917-2923 (1993)) for
mammalian cells or 2 micron or ars plasmids for yeast (Strathern et
al., The Molecular Biology of the Yeast Saccaromyces, CSH Press
(1982)) are used. Integrative vectors such as pJM 103, pJM 113 or
pSGMU2 are preferred for B. subtilis (Perego, Chap. 42, pp. 615-624
in: Bacillus subtilis and Other Gram-Positive Bacteria, A.
Sonenshein, J. Hoch, and R. Losick, eds., 1993).
[0266] For example, an efficiently secreted thermostable DNA
polymerase can be evolved, thus allowing the performance of DNA
polymerization assays with little or no purification of the
expressed DNA polymerase. Such a procedure would be preferred for
the expression of libraries of mutants of any protein that one
wished to test in a high throughput assay, for example any of the
pharmaceutical proteins listed in Table I, or any industrial
enzyme. Initial constructs are made by fusing a signal peptide such
as that from STII or OmpA to the amino terminus of the protein to
be secreted. A gene cluster of cloned genes believed act in the
secretory pathway of interest are mutagenized and coexpressed with
the target construct. Individual clones are screened for expresion
of the gene product. The secretory gene clusters from improved
clones are recovered and recloned and introduced back into the
original host. Preferably, they are first subjected to mutagenesis
before the process is repeated. This cycle is repeated until the
desired improvement in expression of secreted protein is
achieved.
[0267] IV. Evolved Polypeptide Properties
[0268] A. Evolved Transition State Analog and Substrate Binding
[0269] There are many enzymes of industrial interest that have
substantially suboptimal activity on the substrate of interest. In
many of these cases, the enzyme obtained from nature is required to
work either under conditions that are very different from the
conditions under which it evolved or to have activity towards a
substrate that is different from the natural substrate.
[0270] The application of evolutionary technologies to industrial
enzymes is often significantly limited by the types of selections
that can be applied and the modest numbers of mutants that can be
surveyed in screens. Selection of enzymes or catalytic antibodies,
expressed in a display format, for binding to transition state
analogs (McCafferty et al., Appl. Biochem. Biotechnol. 47:157-171
(1994)) or substrate analogs (Janda et al., Proc. Natl. Acad. Sci.
(U.S.A.) 91:2532-2536, (1994)) represents a general strategy for
selecting for mutants with with improved catalytic efficiency.
[0271] Phage display O'Neil et al., Current Biology 5:443-449
(1995) and the other display formats Gates et al., J. Mol. Biol.
255:373-386 (1995); Mattheakis et al., Proc. Natl. Acad. Sci.
(U.S.A.) 91:9022-9026 1994 described herein represent general
methodologies for applying affinity-based selections to proteins of
interest. For example, Matthew and Wells (Science 260:1113-1117
(1993)) have used phage display of a protease substrate to select
improved substrates. Display of active enzymes on the surface of
phage, on the other hand, allows selection of mutant proteins with
improved transition state analog binding. Improvements in affinity
for transition state analogs correlate with improvements in
catalytic efficiency. For example, Patten et al., Science
271:1086-1091 (1996) have shown that improvements in affinity of a
catalytic antibody for its hapten are well correlated with
improvements in catalytic efficiency, with an 80-fold improvement
in kcat/Km being achieved for an esterolytic antibody.
[0272] For example, an enzyme used in antibiotic biosynthesis can
be evolved for new substrate specificity and activity under desired
conditions using phage display selections. Some antibiotics are
currently made by chemical modifications of biologically produced
starting compounds. Complete biosynthesis of the desired molecules
is currently impractical because of the lack of an enzyme with the
required enzymatic activity and substrate specificity (Skatrud,
TIBTECH 10:324-329, September 1992). For example,
7-aminodeacetooxycephalosporanic acid (7-ADCA) is a precursor for
semi-synthetically produced cephalosporins. 7-ADCA is made by a
chemical ring expansion of penicillin G followed by enzymatic
deacylation of the phenoxyacetal group. 7-ADCA can be made
enzymatically from deacetylcephalosporin C (DAOC V), which could in
turn be derived from penicillin V by enzymatic ring expansion if a
suitably modified penicillin expandase could be evolved (Cantwell
et al., Curr. Genet. 17:213-221 (1990),. Thus, 7-ADCA could in
principle be produced enzymatically from penicillin V using a
modified penicillin N expandase, such as mutant forms of the S.
clavuligerus cefE gene (Skatrud, TIBTECH 10:324-329, September
1992). However, penicillin V is not accepted as a substrate by any
known expandase with sufficient efficiency to be commercially
useful. As outlined below RSR techniques of the invention can be
used to evolve the penicillin expandase encoded by cefE or other
expandases so that they will use penicillin V as a substrate.
[0273] Phage display or other display format selections are applied
to this problem by expressing libraries of cefE penicillin
expandase mutants in a display format, selecting for binding to
substrates or transition state analogs, and applying RSR to rapidly
evolve high affinity binders. Candidates are further screened to
identify mutants with improved enzymatic activity on penicillin V
under desired reaction conditions, such as pH, temperature, solvent
concentration, etc. RSR is applied to further evolve mutants with
the desired expandase activity. A number of transition state
analogs (TSA's) are suitable for this reaction. The following
structure is the initial TSA that is used for selection of the
display library of cefE mutants: 1
[0274] Libraries of the known penicillin expandases (Skatrud,
TIBTECH 10:324-329(1992); Cantwell et al., Curr. Genet. 17:13-221
(1990)) are made as described herein. The display library is
subjected to selection for binding to penicillin V and/or to
transition state analog given above for the conversion of
penicillin V to DAOC V. These binding selections may be performed
under non-physiological reaction conditions, such as elevated
temperature, to obtain mutants that are active under the new
conditions. RSR is applied to evolve mutants with 2-10.sup.5 fold
improvement in binding affinity for the selecting ligand. When the
desired level of improved binding has been obtained, candidate
mutants are expressed in a high throughput format and specific
activity for expanding penicillin V to DAOC V is quantitatively
measured. Recombinants with improved enzymatic activity are
mutagenized and the process repeated to further evolve them,
[0275] Retention of TSA binding by a displayed enzyme (e.g., phage
display, lac headpiece dimer, polysome display, etc.) is a good
selection for retention of the overall integrity of the active site
and hence can be exploited to select for mutants which retain
activity under conditions of interest. Such conditions include but
are not limited to: different pH optima, broader pH optima,
activity in altered solvents such as DMSO (Seto et al., DNA
Sequence 5:131-140 (1995)) or formamide (Chen et al., Proc. Natl.
Acad. Sci. (U.S.A.) 90:5618-5622, (1993)) altered temperature,
improved shelf life, altered or broadened substrate specificity, or
protease resistance. A further example, the evolution of a
p-nitrophenyl esterase, using a mammalian display format, is
provided below.
[0276] B. Improvement of DNA and RNA Polymerases
[0277] Of particular commercial importance are improved polymerases
for use in nucleic acid sequencing and polymerase chain reactions.
The following properties are attractive candidates for improvement
of a DNA sequencing polymerase: (1) suppression of termination by
inosine in labelled primer format (H. Dierick et al., Nucleic Acids
Res. 21:4427-4428 (1993)) (2) more normalized peak heights,
especially with fluorescently labelled dideoxy terminators (Parker
et al., BioTechniques 19:115-121 (1995)), (3) better sequencing of
high GC content DNA (>60% GC) by, for example, tolerating
>10% DMSO (D. Seto et al., DNA Sequence 5:131-140 (1995);
Scheidl et al., BioTechniques 19(5):691-694 (1995)), or (4)
improved acceptance of novel base analogs such as inosine, 7-deaza
dGTP (Dierick et al., Nucleic Acids Res. 21:4427-4428 (1993)) or
other novel base analogs that improve the above properties.
[0278] Novel sequencing formats have been described which use
matrix assisted laser desorption ionization time of flight
(MALDT-TOF) mass spectroscopy to resolve dideoxy ladders (Smith,
Nature Biotechnology 14:1084-1085 (1996)). It is noted in Smith's
recent review that fragmentation of the DNA is the singular feature
limiting the development of this method as a viable alternative to
standard gel electrophoresis for DNA sequencing. Base analogs which
stabilize the N-glycosidic bond by modifications of the purine
bases to 7-deaza analogs (Kirpekar et al., Rapid Comm. in Mass
Spec. 9:525-531 (1995)) or of the 2' hydroxyl (such as 2'-H or
2'-F) "relieve greatly the mass range limitation" of this technique
(Smith, 1996). Thus, evolved polymerases that can efficiently
incorporate these and other base analogs conferring resistance to
fragmentation under MALDI-TOF conditions are valuable
innovations.
[0279] Other polymerase properties of interest for improvement by
RSR are low fidelity thermostable DNA polymerase for more efficient
mutagenesis or as a useful correlate for acceptance of base analogs
for the purposes described above; higher fidelity polymerase for
PCR (Lundberg et al., Gene 108:1-6 (1991)); higher fidelity reverse
transcriptase for retroviral gene therapy vehicles to reduce
mutation of the therapeutic construct and of the retrovirus;
improved PCR of GC rich DNA and PCR with modified bases (S. Turner
and F. J. Jenkins, BioTechniques 19(1) :48-52 (1995)).
[0280] Thus, in some embodiments of the invention, libraries of
mutant polymerase genes are screened by direct high throughput
screening for improved sequencing properties. The best candidates
are then subjected to RSR. Briefly, mutant libraries of candidate
polymerases such as Taq polymerase are constructed using standard
methods such as PCR mutagenesis (Caldwell et al., PCR Meth. App.
2:28-33 (1992)) and/or cassette mutagenesis (Sambrook et al.,
Molecular Cloning, CSH Press (1987)). Incorporation of mutations
into Taq DNA polymerase such as the active site residue from T7
polymerase that improves acceptance of dideoxy nucleotides (Tabor
and Richardson, J. Biol. Chem . 265:8322-8328 1990); and mutations
that inactivate the 5'-3' exonuclease activity R. S. Rano,
BioTechniques 18:390-396 1995 are incorporated into these
libraries. The reassembly PCR technique, for example, as described
above is especially suitable for this problem. Similarly, chimeric
polymerase libraries are made by breeding existing thermophilic
polymerases, sequenase, and E. coli polI with each other using the
bridge oligonucleotide methods describes above. The libraries are
expressed in formats wherein human or robotic colony picking is
used to replica pick individual colonies into 96 well plates where
small cultures are grown, and polymerase expression is induced.
[0281] A high throughput, small scale simple purification for
polymerase expressed in each well is performed. For example, simple
single-step purifications of His-tagged Taq expressed in E. coli
have been described (Smirnov et al., Russian J. Bioorganic Chem.
21(5):341-342 (1995)), and could readily be adapted for a 96-well
expression and purification format.
[0282] A high throughput sequencing assay is used to perform
sequencing reactions with the purified samples. The data is
analyzed to identify mutants with improved sequencing properties,
according to any of these criteria: higher quality ladders on
GC-rich templates, especially greater than 60% GC, including such
points as fewer artifactual termination products and stronger
signals than given with the wild-type enzyme; less termination of
reactions by inosine in primer Labelled reactions, e.g.,
fluorescent labelled primers; less variation in incorporation of
signals in reactions with fluorescent dideoxy nucleotides at any
given position; longer sequencing ladders than obtained with the
wild-type enzyme, such as about 20 to 100 nucleotides; improved
acceptance of other known base analogs such as 7-deaza purines;
improved acceptance of new base analogs from combinatorial
chemistry libraries (See, for example, Hogan, Nature 384
(Supp):17-1996).
[0283] The best candidates are then subjected to mutagenesis, and
then selected or screened for the improved sequencing properties
described above.
[0284] In another embodiment, a screen or selection is performed as
follows. The replication of a plasmid can be placed under obligate
control of a polymerase expressed in E. coli or another
microorganism. The effectiveness of this system has been polymerase
beta (Sweasy et al., Proc. Natl. Acad. Sci. (U.S.A.) 90:4626-4630,
(1993)), Taq polymerase (Suzuki et al., Proc. Natl. Acad. Sci.
(U.S.A.) 93:9670-9675 (1996)), or HIV reverse transcriptase (Kim et
al., Proc. Natl. Acad. Sci. (U.S.A.) 92:684-688 (1995) ) . The
mutant polymerase gene is placed on a plasmid bearing a colE1
origin and expressed under the control of an arabinose promoter.
The library is enriched for active polymerases essentially as
described by Suzuki et al., (supra), with polymerase expression
being induced by the presence of arabinose in the culture.
[0285] A further quantitative screen utilizes the presence of GFP
(green fluorescence protein) on the same plasmid, replica plating
onto arabinose at the nonpermissive temperature in the absence of a
selective antibiotic, and using a fluorimeter to quantitatively
measure fluorescence of each culture. GFP activity correlates with
plasmid stability and copy number which is in turn dependent on
expression of active polymerase.
[0286] A polymerase with a very high error rate would be a superior
sequencing enzyme, as it would have a more normalized signal for
incorporation of base analogs such as the currently used
fluorescently labelled dideoxies because it will have reduced
specificity and selectivity. The error rates of currently used
polymerases are on the order of 10.sup.-5 to 10.sup.-6, orders of
magnitude lower than what can be detected given the resolving power
of the gel systems. An error rate of 1%, and possibly as high as
lo, could not be detected by current gel systems, and thus there is
a large window of opportunity to increase the "sloppiness" of the
enzyme. An error-prone cycling polymerase would have other uses
such as for hypermutagenesis of genes by PCR.
[0287] In some embodiments, the system described by Suzuki (Suzuki
et al., Proc. Natl. Acad. Sci. (U.S.A.) 96:9670-9675 (1996) is used
to make replication of a reporter plasmid dependent on the
expressed polymerase. This system puts replication of the first
220-300 cases next to the ColE1 origin directly under the control
of the expressed polymerase (Sweasy and Loeb, J. Bact.
177:2923-2925 1995); Sweasy et al., Proc. Natl. Acad. Sci. (U.S.A.)
90: 4626-4630 1993. A screenable or selectable reporter gene
containing stop codons is positioned in this region, such as LacZ
alpha containing one, two or three stop codons. The constructs are
grown on arabinose at the nonpermissible temperature, allowed to
recover, and plated on selective lactose minimal media that demands
reversion of the codons in the reporter cassette. Mutant
polymerases are recovers from the survivors by PCR. The survivors
are enriched for mutators because their mutator phenotype increases
the rate of reversion of stop codons in the reporter lacZ alpha
fragment
[0288] The polymerase genes from the survivors are subjected to
RSR, then the polymerase mutants are retransformed into the
indicator strain. Mutators can be visually screened by plating on
arabinose/Xgal plates at the nonpermissive temperature. Mutator
polymerases will give rise to colonies with a high frequency of
blue papillae due to reversion of the stop codon(s). Candidate
papillators can be rescreened by picking a non-papillating region
of the most heavily papillated colonies (i.e, "best" colonies) and
replating on the arabinose/Xgal indicator medium to further screen
for colonies with increased papillation rates. These steps are
repeated until a desired reversion rate is achieved (e.g.,
10.sup.-2 to 10.sup.-3 mutations per base pair per
replication).
[0289] Colonies which exhibit high frequency papillation are
candidates for encoding an error prone polymerase. These candidates
are screened for improved sequencing properties essentially as for
the high throughput screen described above. Briefly, mutant Taq
proteins are expressed and purified in a 96-well format. The
purified proteins are used in sequencing reactions and the sequence
data are analyzed to identify mutants that exhibit the improvements
outlined herein. Mutants with improved properties are subjected to
RSR and rescreened for further improvements in function.
[0290] In some embodiments, GFP containing stop condons instead of
lacZ alpha with stop codons is used for the construction. Cells
with reverted stop codons in GFP are selected by fluorescence
activated cell sorter (FACS). In general, FACS selection is
performed by gating the brightest about 0.1-10%, preferably the top
0.1 to 1%, and collected according to a protocol similar to that of
Dangl et al., (Cytometry 2(6):395-401 (1982)). In other
embodiments, the polA gene is flanked with lox sites or other
targets of a site specific recombinase. The recombinase is induced,
thus allowing one to inducibly delete polA gene (Mulbery et al.,
Nucleic Acid Res. 23:485-490 (1995)). This would allow one to
perform "Loeb-type" selections at any temperature and in any host.
For example, one could set up such a selection in a recA deficient
mesophile or thermophile by placing the polA homologue in an
inducibly deletable format and thus apply the selection for active
polymerase under more general conditions.
[0291] In further embodiments, this general system is preferred for
directed in vivo mutagenesis of genes. The target gene is cloned
into the region near a plasmid origin of replication that puts its
replication obligately under control of the error prone polymerase.
The construct is passaged through a polA(ts) recA strain and grown
at the nonpermissive temperature, thus specifically mutagenizing
the target gene while replicating the rest of the plasmid with high
fidelity.
[0292] In other embodiments, selection is based on the ability of
mutant DNA polymerases to PCR amplify DNA under altered conditions
or by utilizing base analogs. The mutant polymerases act on the
template that encodes them in a PCR amplification, thus
differentially replicating those polymerases.
[0293] In brief, an initial library of mutants is replica plated.
Polymerase preparations are done in a 96-well format. Crude plasmid
preparations are made of the same set. Each plasmid prep is
PCR-amplified using the polymerase prep derived from that plasmid
under the conditions for which one wishes to optimize the
polymerase (e.g , added DMSO or formamide, altered temperature of
denaturation or extension, altered buffer salts, PCR with base
analogs such a-thiol dNTP's for use with mass spectroscopy
sequencing, PCR of GC rich DNA (>60% GC),PCR with novel base
analogs such as 7-deaza purines, 2' fluoro dNTP's, rNTP's, PCR with
inosine, etc.). The amplified genes are pooled, cloned, and
subjected to mutagenesis, and the process repeated until an
improvement is achieved.
[0294] C. Evolved Phosphonatase
[0295] Alkaline phosphatase is a widely used reporter enzyme for
ELISA assays, protein fusion assays, and in a secreted form as a
reporter gene for mammalian cells. The chemical lability of
p-nitrophenyl phosphate (pNPP) substrates and the existence of
cellular phosphatases that cross-react with pNPP is an important
limitation on the sensitivity of assays using this reporter gene. A
reporter gene with superior signal to noise properties can be
developed based on hydrolysis of p-nitrophenyl phosphonates, which
are far more stable to base catalyzed hydrolysis than the
corresponding phosphates. Additionally, there are far fewer
naturally occurring cellular phosphonatases than alkaline
phosphatases. Thus a p-nitrophenyl phosphonatase is an attractive
replacement for alkaline phosphatase because the background due to
chemical and enzymatic hydrolysis is much lower. This will allow
one to make ELISA's more sensitive for detecting very small
concentrations of antigen.
[0296] Chen et al. (J. Mol. Biol. 234:165-178 (1993)) have shown
that a Staph. aureus beta-lactamase can hydrolyze p-nitrophenyl
phosphonate esters with single turnover kinetics. The active site
Ser70 (the active site nucleophile for beta lactam hydrolysis)
forms a covalent intermediate with the substrate. This is analogous
to the first step in hydrolysis of beta lactams, and this enzyme
can be evolved by RSR to hydrolyze phosphonates by a mechanism
analogous to beta lactam hydrolysis. Metcalf and Wanner have
described a cryptic phosphonate utilizing operon (phn) in E. coli,
and have constructed strains bearing deletions of the phn operon
(J. Bact. 175:3430-3442 (1993)). This paper discloses selections
for growth of E. coli on phosphate free minimal media where the
phosphorous is derived from hydrolysis of alkyl phosphonates by
genes in the phn operon. Thus, one could select for evolved
p-nitrophenyl phosphonatases that are active using biochemical
selections on defined minimal media. Specifically, an efficient
phosphonatase is evolved as follows. A library of mutants of the
Staph. aureus beta lactamase or one of the E. coli phn enzymes is
constructed. The library is transformed into E. coli mutants
wherein the phn operon has been deleted, and selected for growth on
phosphate free MOPS minimal media containing p-nitrophenyl
phosphonate. RSR is applied to selected mutants to further evolve
the enzyme for improved hydrolysis of p-nitrophenyl
phosphonates.
[0297] D. Evolved Detergent Proteases
[0298] Proteases and lipases are added in large quantities to
detergents to enzymatically degrade protein and lipid stains on
clothes. The incorporation of these enzymes into detergents has
significantly reduced the need for surfactants in detergents will a
consequent reduction in the cost of formulation of detergents and
improvement in stain removal properties. Proteases with improved
specific activity, improved range of protein substrate specificity,
improved shelf life, improved stability at elevated temperature,
and reduced requirements for surfactants would add value to these
products.
[0299] As an example, subtilisin can be evolved as follows. The
cloned subtilisin gene (von der Osten et al., J. Biotechnol.
28:55-68 (1993)) can be subjected to RSR using growth selections on
complex protein media by virtue of secreted subtilisin degrading
the complex protein mixture. More specifically, libraries of
subtilisin mutants are constructed in an expression vector which
directs the mutant protein to be secreted by Bacillus subtilus.
Bacillus hosts transformed with the libraries are grown in minimal
media with complex protein formulation as carbon and/or nitrogen
source. Subtilisin genes are recovered from fast growers and
subjected to RSR, then screened for improvement in a desired
property.
[0300] E. Escape of Phage from a "Protein Net"
[0301] In some embodiments, selection for improved proteases is
performed as follows. A library of mutant protease genes is
constructed on a display phage and the phage grown in a multiwell
format or on plates. The phage are overlayed with a "protein net
which ensnares the phage. The net can consist of a protein or
proteins engineered with surface disulphides and then crosslinked
an auxiliary matrix to further trap the phage. The phage are
further incubated, then washed to collect liberated phage wherein
the displayed protease was able to liberate the phage from the
protein net. The protease genes are then subjected to RSR for
further evolution. A further embodiment employs a library of
proteases encoded by but not displayed on a phagemid wherein
streptavidin is fused to pIII by a peptide linker. The library pf
protease mutants is evolved to cleave the linker by selecting
phagemids on a biotin column between rounds of amplification.
[0302] In a further embodiment, the protease is not necessarily
provided in a display format. The host cells secrete the protease
encoded by but not surface diplayed by a phagemid, while
constrained to a well, for example, in a microtiter plate. Phage
display format is preferred where an entire high titre lysate is
encased in a protein net matrix, and the phage expressing active
and broad specificity proteases digesting the matrix to be
liberated for the next round of amplification, mutagenesis, and
selection.
[0303] In a further embodiment, the phage are not constrained to a
well but, rather, protein binding filters are used to make a colony
of plaque lifts and are screened for activity with chromogenic or
fluorogenic substrates. Colonies or plaques corresponding to
positive spots on the filters are picked and the encoded protease
genes are recovered by, for example, PCR. The protease genes are
then subjected to RSR for further evolution.
[0304] F. Screens for Improved Protease Activity
[0305] Peptide substrates containing fluoropores attached to the
carboxy terminus and fluorescence quenching moities on the amino
terminus, such as those described by Holskin, et al, (Anal.
Biochem. 227:148-55 (1995)) (e g., 4-4'-dimethylaminophenazo,
benzoyl-arg-gly-val-val-asn-ala-ser-ser-
-arg-leu-ala-5-(2'-aminoethyl-amino)-naphthalene-1-sulfonic acid)
are used to screen protease mutants for broadened or altered
specificity. In brief, a library of peptide substrates is designed
with a flourophore on th amino terminus and a potent fluorescence
quencher on He carboxy terminus, or vice versa. Supernatants
containing secretes proteases are incubated either separately with
various members of the library or with a complex cocktail. Those
proteases which are highly active and have broad specificity will
cleave the majority of the peptides, thus releasing the fluorophore
from the quencher and giving a positive signal on a fluorimeter.
This technique is amenable to a high density multiwell format.
[0306] G. Improving Pharmaceutical Proteins Using RSR
[0307] Table I lists proteins that are of particular commercial
interest to the pharmaceutical industry. These proteins are all
candidates for RSR evolution to improve function, such as ligand
binding, shelf life, reduction of side effects through enhanced
specificity, etc. All are well-suited to manipulation by the
techniques of the invention. Additional embodiments especially
applicable to this list are described below.
[0308] First, high throughput methods for expressing and purifying
libraries of mutant proteins, similar to the methods described
above for Taq polymerase, are applied to the proteins of Table I.
These mutants are screened for activity in a functional assay. For
example, mutants of IL2 are screened for resistance to plasma or
tissue proteases with retention of activity for the low affinity
IL2 receptor but with loss of activity on the high affinity IL2
receptor. The genes from mutants with improved activity relative to
wild-type are recovered, and subjected to RSPR to improve the
phenotype further.
[0309] Preferably, the libraries are generated in a display format
such that the mature folded protein is physically linked the
genetic information that encodes it. Examples include phage display
using filamentous phage (O'Neil et al., Current Biology 5:443-449
(1995)) or bacteriophage lambda gene V display (Dunn, J. Mol. Biol.
248:497-506 1995), peptides on plasmids Gates et al., J. Mol. Biol.
255:373-386 1995 where the polypeptide of interest is fused to a
lac headpiece dimer and the nascent translation product binds to a
site encoded on the plasmid or PCR product, and polysome display
Mattheakis et al., Proc. Natl. Acad. Sci. U.S.A. 91:9022-9026
1994)) where ribosomes are stalled on mRNA molecules such that the
nascent polypeptide is exposed for interaction with cognate ligands
without disrupting the stalled ribosome/mRNA complex. Selected
complexes are subjected to RT-PCR to recover the genes.
[0310] When so displayed, affinity binding of the recombinant phage
is often done using a receptor for the protein of interest. In some
cases it is impractical to obtain purified receptor with retention
of all desired biological characteristics (for example,
7-transmembrane (7-TM) receptors. In such cases, one could use
cells expressing the receptor as the panning substrate. For
example, Barry et al. (Nat. Med. 2:299-305 (1996)) have described
successful panning of M13 libraries against whole cells to obtain
phage that bind to the cells expressing a receptor of interest.
This format could be generally applied to any of the proteins
listed in Table I.
[0311] In some embodiments, the following method can be used for
selection. A lysate of phage encoding IFN alpha mutants, for
example, can be used directly at suitable dilution to stimulate
cells with a GFP reporter construct (Crameri et al., Nat. Med.
14:315-319 (1996)) under the control of an IFN responsive promoter,
such as an MHC class I promoter. Phage remaining attached after
stimulation, expression and FACS purification of the responsive
cells, can be purified by FACS. Preferably, the brightest cells are
collected. The phage are collected and their DNA subjected to RSR
until the level of desired improvement is achieved.
[0312] Thus, for example, IL-3 is prepared in one of these display
formats and subjected to RSR to evolve an agonist with a desired
level of activity. A library of IL3 mutants on a filamentous phage
vector is created and affinity selected ("panned") against purified
IL3 receptor to obtain mutants with improved affinity. The mutant
IL-3 genes are recovered by PCR, subjected to RSR, and recloned
into the display vector. The cycle is repeated until the desired
affinity or agonist activity is achieved.
[0313] Many proteins of interest are expressed as dimers or higher
order multimeric forms. In some embodiments, the display formats
descibed above preferentially are applied to a single chain version
of the protein. Mutagenesis, such as RSR, can be used in these
display formats to evolve improved single chain derivatives of
multimeric factors which initially have low but detectable
activity. This strategy is described in more detail below.
[0314] H. Whole Cell Selections
[0315] In some embodiments, the eukaryotic cell is the unit of
biological selection. The following general protocol can be used to
apply RSR to the improvement of proteins using eukaryotic cells as
the unit of selection: (1) transfection of libraries of mutants
into a suitable host cell, (2) expression of the encoded gene
product(s) either transiently or stably, (3) functional selection
for cells with an improved phenotype (expression of a receptor with
improved affinity for a target ligand; viral resistance, etc., (4)
recovery of the mutant genes by, for example, PCR followed by
preparation of HIRT supernatants with subsequent tranformation of
E. coli, (5) RSR and (6) repetition of steps (1)-(5) until the
desired degree of improvement is achieved.
[0316] For example, previous work has shown that one can use
mammalian surface display to functionally select cells expressing
cloned genes, such as using an antibody to clone the gene for an
expressed surface protein (Reviewed by Seed, Curr. Opin.
Biotechnol. 6:567-573 (1995)). Briefly, cells are transiently
transfected with libraries of cloned genes residing on replicating
episomal vectors. An antibody directed against the protein of
interest (whose gene one wishes to clone) is immobilized on a solid
surface such as a plastic dish, and the transfected cells
expressing the protein of interest are affinity selected.
[0317] For example, the affinity of an antibody for a ligand can be
improved using mammalian surface display and RSR. Antibodies with
higher affinity for their cognate ligands are then screened for
improvement of one or more of the following properties: (1)
improved therapeutic properties increased cell killing,
neutralization of ligands, activation of signal transduction
pathways by crosslinking receptors), (2) improved in vivo imaging
applications (detection of the antibody by covalent/noncovalent
binding of a radionuclide or any agent detectable outside of the
body by noninvasive means, such as NMR), (3) improved analytical
applications (ELISA detection of proteins or small molecules), and
(4) improved catalysts (catalytic antibodies). The methods
described are general and can be extended to any receptor-ligand
pair of interest. A specific example is provided in the
experimental section.
[0318] The use of a one mutant sequence-one transfected cell
protocol is a preferred design feature for RSR based protocols
because the point is to use functional selection to identify
mutants with improved phenotypes and, if the transfection is not
done in a "clonal" fashion, the functional phenotype of any given
cell is the result of the sum of many transfected sequences.
Protoplast fusion is one method to achieve this end, since each
protoplast contains typically greater than 50 copies each of a
single plasmid variant. However, it is a relatively low efficiency
process (about 10.sup.3-10.sup.4 transfectants), and it does not
work well on some non-adherent cell lines such as B cell lines.
Retroviral vectors provide a second alternative, but they are
limited in the size of acceptable insert (<10 kb) and
consistent, high expression levels are sometimes difficult to
achieve. Random integration results in varying expression levels,
thus introducing noise and limiting one's ability to distinguish
between improvements in the affinity of the mutant protein vs.
increased expression. A related class of strategies that can be
used effectively to achieve "one gene-one cell" DNA transfer and
consistent expression levels for RSR is to use a viral vector which
contains a lox site and to introduce this into a host that
expresses ore recombinase, preferably transiently, and contains one
or more lox sites integrated into its genome, thus limiting the
variability of integration sites (Rohlman et al. Nature Biotech.
14:1562-1565 (1996)).
[0319] An alternative strategy is to transfect with limiting
concentrations of plasmid (i.e., about one copy per cell) using a
vector that can replicate in the target cells, such as is the case
with plasmids bearing SV40 origins transfected into COS cells. This
strategy requires that either the host cell or the vector supply a
replication factor such as SV40 large T antigen. Northrup et al.
(J. Biol. Chem. 268:2917-2923 (1993)) describe a strategy wherein a
stable transfectant expressing SV40 large T antigen is then
transfected with vectors bearing SV40 origins. This format gave
consistently higher transient expression and demonstrable plasmid
replication, as assayed by sensitivity to digestion by Dpn I.
Transient expression (i.e, non-integrating plasmids) is a preferred
format for cellular display selections because it reduces the cycle
time and increases the number of mutants that can be screened.
[0320] The expression of SV40 large T antigen or other replication
factors may have deleterious effects on or may work inefficiently
in some cells. In such cases, RSR is applied to the replication
factor itself to evolve mutants with improved activity in the cell
type of interest. A generic protocol for evolving such a factor is
as follows:
[0321] The target cell is transfected with GFP cloned onto a vector
containing SV40 large T antigen, an SV40 origin, and a reporter
gene such as GFP; a related format is cotransfection with limiting
amounts of the SV40 large T antigen expression vector and an excess
of a reporter such as GFP cloned onto an SV40 origin containing
plasmid. Typically after 1-10 days of transient expression, the
brightest cells are purified by FACS. SV40 large T antigen mutants
are recovered by PCR, and subjected to mutagenesis. The cycle is
repeated until the desired level of improvement is obtained.
[0322] I. Autocrine Selection
[0323] In some embodiments, mutant proteins are selected or
screened based on their ability to exert a biological effect in an
autocrine fashion on the cell expressing the mutant protein. For
example, a library of alpha interferon genes can be selected for
induction of more potent or more specific antiviral activity as
follows. A library of interferon alpha mutants is generated in a
vector which allows for induction of expression (i.e. under control
of a metallothionein promoter) and efficient secretion in a
multiwell format (96-well for example) with one or a few
independent clones per well. In some embodiments, the promoter is
not inducible, and may be constitutive.
[0324] Expression of the cloned interferon genes is induced. The
cells are challenged with a cytotoxic virus against which one
wishes to evolve an optimized interferon (for example vesicular
stomatitus virus or HIV). Surviving cells are recovered. The cloned
interferon genes are recovered by PCR amplification, subjected to
RSR, and cloned back into the transfection vector and retransfected
into the host cells. These steps are repeated until the desired
level of antiviral activity is evolved.
[0325] In some embodiments, the virus of interest is not strongly
cytotoxic. In this case a conditionally lethal gene, such as herpes
simplex virus thymidine kinase, is cloned into the virus and after
challenge with virus and recovery, conditionally lethal selective
conditions are applied to kill cells that are infected with virus.
An example of a conditionally lethal gene is herpes TK, which
becomes lethal upon treating cells expressing this gene with the
thymidine analog acyclovir. In some embodiments, the
antiproliferative activity of the cloned interferons is selected by
treating the cells with agents that kill dividing cells (for
example, DNA alkylating agents).
[0326] In some embodiments, potent cytokines are selected by
expressing and secreting a library of cytokines in cells that have
GFP or another reporter under control of a promoter that is induced
by the cytokine, such as the MHC class I promoter being induced by
evolved variants of alpha interferon. The signal transduction
pathway is configured such that the wild type cytokine to be
evolved gives a weak but detectable signal.
[0327] J. Half Life in Serum
[0328] In some embodiments of the invention, proteins are evolved
by RSR to have improved half life in serum. A preferred method for
improving half-life is evolving the affinity of a protein of
interest for a long lived serum protein, such as an antibody or
other abundant serum protein. Examples of how affinity for an
antibody can enhance serum half life include the co-administration
of IL2 and anti-IL2 antibodies which increases serum half-life and
anti-tumor activity of human recombinant IL2 (Courtney et al.,
Immunopharmacology 28:223-232 (1994)).
[0329] The eight most abundant human serum proteins are serum
albumin, immunoglobulins, lipoproteins, haptoglobin, fibrinogen,
transferrin, alpha-1 antitrypsin, and alpha-2 macroglobulin
(Doolittle, chapter 6, The Plasma Proteins F. Putnam, ed.; Academic
Press, 1984). These and other abundant serum proteins such as
ceruloplasmin and fibronectin are the primary targets against which
to evolve binding sites on therapeutic proteins such as in Table I
for the purpose of extending half-life. In the case of antibodies,
the preferred strategy is to evolve affinity for constant regions
rather than variable regions in order to minimize individual
variation in the concentration of the relevant target epitope
(antibody V region usage between different individuals is
significantly variable).
[0330] Binding sites of the desired affinity are evolved by
applying phage display, peptides on plasmid display or polysome
display selections to the protein of interest. One could either
mutagenize an existing binding site or otherwise defined region of
the target protein, or append a peptide library to the N terminus,
C terminus, or internally as a functionally nondisruptive loop.
[0331] In other embodiments of the invention, half life is improved
by derivatization with PEG, other polymer conjugates or half-life
extending chemical moieties. These are established methods for
extending half-life of therapeutic proteins (R. Duncan, Clin.
Pharmacokinet 27:290-306 (1994); Smith et al., TIBTECH 11 397-403
(1993)) and can have the added benefit of reducing immunogenicity
(R. Duncan, Clin. Pharmacokinet 27:290-306 (1994)). However,
derivatization can also result in reduced affinity of the
therapeutic protein for its receptor or ligand. RSR is used to
discover alternative sites in the primary sequence that can be
substituted with lysine or other appropriate residues for chemical
or enzymatic conjugation with half-life extending chemical
moieties, and which result in proteins with maximal retention of
biological activity.
[0332] A preferred strategy is to express a library of mutants of
the protein in a display format, derivatize the library with the
agent of interest (i.e. PEG) using chemistry that does not
biologically inactivate the display system, select based on
affinity for the cognate receptor, PCR amplify the genes encoding
the selected mutants, shuffle, reassemble, reclone into the display
format, and iterate until a mutant with the desired activity post
modification is obtained. An alternative format is to express,
purify and derivatize the mutants in a high throughput format,
screen for mutants with optimized activity, recover the
corresponding genes, subject the genes to RSR and repeat.
[0333] In further embodiments of the invention, binding sites for
target human proteins that are localized in particular tissues of
interest are evolved by RSR. For example, an interferon that
localizes efficiently to the liver can be engineered to contain a
binding site for a liver surface protein such as hepatocyte growth
factor receptor. Analogously, one could evolve affinity for
abundant epitopes on ervthrocytes such as ABO blood antigens to
localize a given protein to the blood stream.
[0334] In further embodiments of the invention, the protein of
interest is evolved to have increased stability to proteases. For
example, the clinical use of IL2 is limited by serious side effects
that are related to the need to administer high doses. High doses
are required due to the short half life (3-5 min, Lotze et al.,
JAMA 256(22):3117-3124 (1986)) and the consequent need for high
doses to maintain a therapeutic level of IL2. One of the factors
contributing to short half-lives of therapeutic proteins is
proteolysis by serum proteases. Cathepsin D, a major renal acid
protease, is responsible for the degradation of IL2 in Balb/c mice
(Ohnishi et al., Cancer Res. 50:1107-1112 (1990)). Furthermore,
Ohnishi showed that treatment of Balb/c mice with pepstatin, a
potent inhibitor of this protease, prolongs the half life of
recombinant human IL2 and augments lymphokine-activated killer cell
activity in this mouse model.
[0335] Thus, evolution of protease resistant variants of IL2 or any
of the proteins listed in Table I that are resistant to serum or
kidney proteases is a preferred strategy for obtaining variant with
extended serum half lives.
[0336] A preferred protocol is as follows. A library of the
mutagenized protein of interest is expressed in a display system
with a gene-distal epitope tag (i.e. on the N-terminus of a phage
display construct such that if it is cleaved off by proteases, the
epitope tag is lost). The expressed proteins are treated with
defined proteases or with complex cocktails such as whole human
serum. Affinity selection with an antibody to the gene distal tag
is performed. A second selection demanding biological function
(e.g., binding to cognate receptor) is performed. Phage retaining
the epitope tag (and hence protease resistant) are recovered and
subjected to RSR. The process is repeated until the desired level
of resistance is attained.
[0337] In other embodiments, the procedure is performed in a
screening format wherein mutant proteins are expressed and purified
in a high throughput format and screened for protease resistance
with retention of biological activity.
[0338] In further embodiments of the invention, the protein of
interest is evolved to have increased shelf life. A library of the
mutagenized nucleic acid squence encoding the protein of interest
is expressed in a display format or high throughput expression
format, and exposed for various lengths of time to conditions for
which one wants to evolve stability (heat, metal ions,
nonphysiological pH of, for example, <6 or >8,
lyophilization, freeze-thawing). Genes are recovered from from
survivors, for example, by PCR. The DNA is subjected to
mutagenesis, such as RSR, and the process repeated until the
desired level of improvement is achieved.
[0339] K. Evolved Single Chain Versions of Multisubunit Factors
[0340] As discussed above, in some embodiments of the invention,
the substrate for evolution by RSR is preferably a single chain
contruction. The possibility of performing asymetric mutagenesis on
constructs of homomultimeric proteins provides important new
pathways for further evolution of such constructs that is not open
to the proteins in their natural homomultimeric states. In
particular, a given mutation in a homomultimer will result in that
change being present in each identical subunit. In single chain
constructs, however, the domains can mutate independently of each
other.
[0341] Conversion of multisubunit proteins to single chain
constructs with new and useful properties has been demonstrated for
a number of proteins. Most notably, antibody heavy and light chain
variable domains have been linked into single chain Fv's (Bird et
al., Science 242:423-426 (1988)), and this strategy has resulted in
antibodies with improved thermal stability (Young et al., FEBS Lett
377:135-139 (1995)), or sensitivity to proteolysis (Solar et al.,
Prot. Eng. 8:717-723 (1995)). A functional single chain version of
IL5, a homodimer, has been constructed, shown to have affinity for
the ILS receptor similar to that of wild type protein, and this
construct has been used to perform assymetric mutagenesis of the
dimer (Li et al., J. Biol. Chem. 271:1817-1820 (1996)). A single
chain version of urokinase-type plasminogen activator has been
made, and it has been shown that the single chain construct is more
resistant to plasminogen activator inhibitor type 1 than the native
homodimer (Higazi et al., Blood 87:3545-3549 (1996)). Finally, a
single-chain insulin-like growth factor I/insulin hybrid has been
constructed and shown to have higher affinity for chimeric
insulin/IGF-1 receptors than that of either natural ligand
(Kristensen et al., Biochem. J. 305:981-986 (1995)).
[0342] In general, a linker is constructed which joins the amino
terminus of one subunit of a protein of interest to the carboxyl
terminus of another subunit in the complex. These fusion proteins
can consist of linked versions of homodimers, homomultimers,
heterodimers or higher order heteromultimers. In the simplest case,
one adds polypeptide linkers between the native termini to be
joined. Two significant variations can be made. First, one can
construct diverse libraries of variations of the wild type sequence
in and around the junctions and in the linkers to facilitate the
construction of active fusion proteins. Secondly, Zhang et al.,
Biochemistry 32:12311-12318 (1993)) have described circular
permutations of T4 lysozyme in which the native amino and carboxyl
termini have been joined and novel amino and carboxyl termini have
been engineered into the protein. The methods of circular
permutation, libraries of linkers, and libraries of junctional
sequences flanking the linkers allow one to construct libraries
that are diverse in topological linkage strategies and in primary
sequence. These libraries are expressed and selected for activity.
Any of the above mentioned strategies for screening or selection
can be used, with phage display being preferable in most cases.
Genes encoding active fusion proteins are recovered, mutagenized,
reselected, and subjected to standard RSR protocols to optimize
their function. Preferably, a population of selected mutant single
chain constructs is PCR amplified in two seprate PCR reactions such
that each of the two domains is amplified separately.
Oligonucleotides are derived from the 5' and 3' ends of the gene
and from both strands of the linker. The separately amplified
domains are shuffled in separate reactions, then the two
populations are recombined using PCR reassembly to generate intact
single chain constructs for further rounds of selection and
evolution.
[0343] V. Improved Properties of Pharmaceutical Proteins
[0344] A. Evolved Specificity for Receptor or Cell Type of
Interest
[0345] The majority of the proteins listed in Table I are either
receptors or ligands of pharmaceutical interest. Many agonists such
as chemokines or interleukins agonize more than one receptor.
Evolved mutants with improved specificity may have reduced side
effects due to their loss of activity on receptors which are
implicated in a particular side effect profile. For most of these
ligand/receptors, mutant forms with improved affinity would have
improved pharmaceutical properties. For example, an antagonistic
form of RANTES with improved affinity for CKR5 should be an
improved inhibitor of HIV infection by virtue of achieving greater
receptor occupancy for a given dose of the drug. Using the
selections and screens outlined above in combination with RSR, the
affinities and specificities of any of the proteins listed in Table
I can be improved. For example the mammalian display format could
be used to evolve TNF receptors with improved affinity for TNF.
[0346] Other examples include evolved interferon alpha variants
that arrest tumor cell proliferation but do not stimulate NK cells,
IL2 variants that stimulate the low affinity IL2 receptor complex
but not the high affinity receptor (or vice versa), superantigens
that stimulate only a subset of the V beta proteins recognized by
the wild type protein (preferably a single V beta), antagonistic
forms of chemokines that specifically antagonize only a receptor of
interest, antibodies with reduced cross-reactivity, and chimeric
factors that specifically activate a particular receptor complex.
As an example of this latter case, one could make chimeras between
IL2 and IL4, 7, 9, or 15 that also can bind the IL2 receptor alpha,
beta and gamma chains (Theze et al., Imm. Today 17:481-486 (1996)),
and select for chimeras that retain binding for the intermediate
affinity IL2 receptor complex on monocytes but have reduced
affinity for the high affinity IL2 alpha, beta, gamma receptor
complex on activated T cells.
[0347] B. Evolved Agonists with Increased Potency
[0348] In some embodiments of the invention, a preferred strategy
is the selection or screening for mutants with increased agonist
activity using the whole cell formats described above, combined
with RSR. For example, a library of mutants of IL3 is expressed in
active form on phage as described by Gram et al. (J. Immun. Meth.
161:169-176 (1993)). Clonal lysates resulting from infection with
plaque-purified phage are prepared in a high through-put format
such as a 96-well microtiter format. An IL3-dependent cell line
expressing a reporter gene such as GFP is stimulated with the phage
lysates in a high throughput 96-well. Phage that result in positive
signals at the greatest dilution of phage supernatants are
recovered; alternatively, DNA encoding the mutant IL3 can be
recovered by PCR. In some embodiments, single cells expressing GFP
under control of an IL3 responsive promoter can stimulated with the
IL3 phage library, and the positive FACS sorted. The nucleic acid
is then subjected to PCR, and the process repeated until the
desired level of improvement is obtained.
1TABLE I POLYPEPTIDE CANDIDATES FOR EVOLUTION Name Alpha-1
antitrypsin Angiostatin Antihemolytic factor Apolipoprotein
Apoprotein Atrial natriuretic factor Atrial natriuretic polypeptide
Atrial peptides C-X-C chemokines (e.g., T39765, NAP-2, ENA-78,
Gro-a, Gro-b, Gro- c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG)
Calcitonin CC chemokines (e.g., Monocyte chemoattractant protein-1,
Monocyte chemoattractant protein-2, Monocyte chemoattractant
protein- 3, Monocyte inflammatory protein-1 alpha, Monocyte
inflammatory protein-1 beta, RANTES, 1309, R83915, R91733, HCC1,
T58847, D31065, T64262) CD40 ligand Collagen Colony stimulating
factor (CSF) Complement factor 5a Complement inhibitor Complement
receptor 1 Factor IX Factor VII Factor VIII Factor X Fibrinogen
Fibronectin Glucocerebrosidase Gonadotropin Hedgehog proteins
(e.g., Sonic, Indian, Desert) Hemoglobin (for blood substitute; for
radiosensitization) Hirudin Human serum albumin Lactoferrin
Luciferase Neurturin Neutrophil inhibitory factor (NIF) Osteogenic
protein Parathyroid hormone Protein A Protein G Relaxin Renin
Salmon calcitonin Salmon growth hormone Soluble complement receptor
I Soluble I-CAM 1 Soluble interleukin receptors (IL-1, 2, 3, 4, 5,
6, 7, 9, 10, 11, 12, 13, 14, 15) Soluble TNF receptor Somatomedin
Somatostatin Somatotropin Streptokinase Superantigens, i.e.,
Staphylococcal enterotoxins (SEA, SEB, SEC1, SEC2, SEC3, SED, SEE),
Toxic shock syndrome toxin (TSST-1) Exfoliating toxins A and B,
Pyrogenic exotoxins A, B, and C, and M. arthritidis mitogen
Superoxide dismutase Thymosin alpha 1 Tissue plasminogen activator
Tumor necrosis factor beta (TNF beta) Tumor necrosis factor
receptor (TNFR) Tumor necrosis factor-alpha TNF alpha)
Urokinase
[0349] C. Evolution of Components of Eukaryotic Signal Transduction
or Transcriptional Pathways
[0350] Using the screens and selections listed above, RSR can be
used in several ways to modify eukaryotic signal transduction or
transcriptional pathways. Any component of a signal transduction
pathway of interest, of the regulatory regions and transcriptional
activators that interact with this region and with chemicals that
induce transcription can be evolved. This generates regulatory
systems in which transcription is activated more potently by the
natural inducer or by analogues of the normal inducer. This
technology is preferred for the development and optimization of
diverse assays of biotechnological interest. For example, dozens of
7 transmembrane receptors (7-TM) are validated targets for drug
discovery (see, for example, Siderovski et al., Curr Biol.,
6(2):211-212 (1996); An et al., FEBS Lett., 375(1-2):121-124
(1995); Raport et al., Gene, 163(2):295-299 (1995); Song et al.,
Genomics, 28(2):347-349 (1995); Strader et al. FASEB J.,
9(9):745-754 (1995); Benka et al., FEBS Lett., 363(1-2):49-52
(1995); Spiegel, J. Clin Endocrinol. Metab., 81(7):2434-2442
(1996); Post et al., FASEB J., 10(7):741-749 (1996); Reisine et
al., Ann NY Acad. Sci., 780:168-175 (1996); Spiegel, Annu. Ref.
Physiol., 58:143-170 (1996); Barak et al., Biochemistry,
34(47):15407-15414 (1995); and Shenker, Baillieres Clin.
Endocrinol. Metab., 9(3):427-451 (1995)). The development of
sensitive high throughput assays for agonists and antagonists of
these receptors is essential for exploiting the full potential of
combinatorial chemistry in discovering such ligands. Additionally,
biodetectors or biosensors for different chemicals can be developed
by evolving 7-TM's to respond agonistically to novel chemicals or
proteins of interest. In this case, selection would be for
contructs that are activated by the new chemical or polypeptide to
be detected. Screening could be done simply with fluorescence or
light activated cell sorting, since the desired improvement is
coupled to light production.
[0351] In addition to detection of small molecules such as
pharmaceutical drugs and environmental pollutants, biosensors can
be developed that will respond to any chemical for which there are
receptors, or for which receptors can be evolved by recursive
sequence recombination, such as hormones, growth factors, metals
and drugs. The receptors may be intracellular and direct activators
of transcription, or they may be membrane bound receptors that
activate transcription of the signal indirectly, for example by a
phosphorylation cascade. They may also not act on transcription at
all, but may produce a signal by some post-transcriptional
modification of a component of the signal generating pathway. These
receptors may also be generated by fusing domains responsible for
binding different ligands with different signalling domains. Again,
recursive sequence recombination can be used to increase the
amplitude of the signal generated to optimize expression and
functioning of chimeric receptors, and to alter the specificity of
the chemicals detected by the receptor.
[0352] For example, G proteins can be evolved to efficiently couple
mammalian 7-TM receptors to yeast signal transduction pathways.
There are 23 presently known G alpha protein loci in mammals which
can be grouped by sequence and functional similarity into four
groups, Gs (Gna, Gna1), Gi (Gnai-2, Gnai-3, Gnai-1, Gnao, Gnat-1,
Gnat-2, Gnaz), Gq (Gnaq, Gna-11, Gna-14, Gna-15) and G12 (Gna-12,
Gna-13) (B. Nurnberg et al., J. Mol. Med., 73:123-132 (1995)). They
possess an endogenous GTP-ase activity allowing reversible
functional coupling between ligand-bound receptors and downstream
effectors such as enzymes and ion channels. G alpha proteins are
complexed noncovalently with G beta and G gamma proteins as well as
to their cognate 7-TM receptor(s). Receptor and signal specificity
are controlled by the particular combination of G alpha, G beta (of
which there are five known loci) and G gamma (seven known loci)
subunits. Activation of the heterotrimeric complex by ligand bound
receptor results in dissociation of the complex into G alpha
monomers and G beta, gamma dimers which then transmit signals by
associating with downstream effector proteins. The G alpha subunit
is believed to be the subunit that contacts the 7-TM, and thus it
is a focal point for the evolution of chimeric or evolved G alpha
subunits that can transmit signals from mammalian 7-TM's to yeast
downstream genes.
[0353] Yeast based bioassays for mammalian receptors will greatly
facilitate the discovery of novel ligands. Kang et al. (Mol. Cell
Biol. 10:2582-2590 (1990)) have described the partial
complementation of yeast strains bearing mutations in SCG1 (GPA1),
a homologue of the alpha subunits of G proteins involved in signal
transduction in mammalian cells, by mammalian and hybrid
yeast/mammalian G alpha proteins. These hybrids have partial
function, such as complementing the growth defect in scg1 strains,
but do not allow mating and hence do not fully complement function
in the pheromone signal transduction pathway. Price et al. (Mol.
Cell Biol. 15:6188-6195 (1995)) have expressed rat somatostatin
receptor subtype 2 (SSTR2) in yeast and demonstrated transmission
of ligand binding signals by this 7-TM receptor through yeast and
chimeric mammalian/yeast G alpha subunits ("coupling") to a HIS3
reporter gene, under control of the pheromone responsive promoter
FUS-1 enabling otherwise HIS3(-) cells to grow on minimal medium
lacking histidine.
[0354] Such strains are useful as reporter strains for mammalian
receptors, but suffer from important limitations as exemplified by
the study of Kang et al., where there appears to be a block in the
transmission of signals from the yeast pheromone receptors to the
mammalian G proteins. In general, to couple a mammalian 7-TM
receptor to yeast signal transduction pathways one couples the
mammalian receptor to yeast, mammalian, or chimeric G alpha
proteins, and these will in turn productively interact with
downstream components in the pathway to induce expression of a
pheromone responsive promoter such as FUS-1. Such functional
reconstitution is commonly referred to as "coupling".
[0355] The methods described herein can be used to evolve the
coupling of mammalian 7-TM receptors to yeast signal transduction
pathways. A typical approach is as follows: (1) clone a 7-TM of
interest into a yeast strain with a modified pheromone response
pathway similar to that described by Price (e.g., strains deficient
in FAR1, a negative regulator of G.sub.1 cyclins, and deficient in
SST2 which causes the cells to be hypersensitive to the presence of
pheromone), (2) construct libraries of chimeras between the
mammalian G alpha protein(s) known or thought to interact with the
GPAL or homologous yeast G alpha proteins, (3) place a selectable
reporter gene such as HIS3 under control of the pheromone
responsive promoter FUS1 (Price et al., Mol. Cell Biol.
15:6188-6195 (1995)). Alternatively, a screenable gene such as
luciferase may be placed under the control of the FUS1 promoter;
(4) transform library (2) into strain (3) (HIS(-)), (5) screen or
select for expression of the reporter in response to the ligand of
interest, for example by growing the library of transformants on
minimal plates in the presence of ligand to demand HIS3 expression,
(6) recover the selected cells, and and apply RSR to evolve
improved expression of the reporter under the control of the
pheromone responsive promoter FUS1.
[0356] A second important consideration in evolving strains with
optimized reporter constructs for signal transduction pathways of
interest is optimizing the signal to noise ratio (the ratio of gene
expression under inducing vs noninducing conditions). Many 7-TM
pathways are leaky such that the maximal induction of a typical
reporter gene is 5 to 10-fold over background. This range of signal
to noise may be insufficient to detect small effects in many high
through put assays. Therefore, it is of interest to couple the 7-TM
pathway to a second nonlinear amplification system that is tuned to
be below but near the threshold of activation in the uninduced
state. An example of a nonlinear amplification system is expression
of genes driven by the lambda P.sub.L promoter. Complex cooperative
interactions between lambda repressor bound at three adjacent sites
in the cI promoter result in very efficient repression above a
certain concentration of repressor. Below a critical threshold
dramatic induction is seen and there is a window within which a
small decrease in repressor concentration leads to a large increase
in gene expression (Ptashne, A Genetic Switch:Phage Lambda and
Higher Organisms, Blackwell Scientific Publ. Cambridge, Mass.,
1992). Analogous effects are seen for some eukaryotic promoters
such as those regulated by GAL4. Placing :he expression of a
limiting component of a transcription factor for such a promoter
(GAL4) under the control of a GAL4 enhanced 7-TM responsive
promoter results in small levels of induction of the 7-TM pathway
signal being amplified to a much larger change in the expression of
a reporter construct also under the control of a GAL4 dependent
promoter.
[0357] An example of such a coupled system is to place GAL4 under
control of the FUS-1 pheromone responsive promoter and to have the
intracellular GAL4 (itself a transcriptional enhancer) level
positively feedback on itself by placing a GAL4 binding site
upstream of the FUS-1 promoter. A reporter gene is also put under
the control of a GAL4 activated promoter. This system is designed
so that GAL4 expression will nonlinearly self-amplify and
co-amplify expression of a reporter gene such as luciferase upon
reaching a certain threshold in the cell. RSR can be used to great
advantage to evolve reporter constructs with the desired signaling
properties, as follows: (1) A single plasmid construct is made
which contains both the GAL4/pheromone pathway regulated GAL4 gene
and the GAL4 regulated reporter gene. (2) This construct is
mutagenized and transformed into the appropriately engineered yeast
strain expressing a 7-TM and chimeric yeast/mammalian protein of
interest. (3) Cells are stimulated with agonists and screened (or
selected) based on the activity of the reporter gene. In a
preferred format, luciferase is the reporter gene and activity is
quantitated before and after stimulation with the agonist, thus
allowing for a quantitative measurement of signal to noise for each
colony. (4) Cells with improved reporter properties are recovered,
the constructs are shuffled, and RSR is applied to further evolve
the plasmid to give optimal signal noise characteristics.
[0358] These approaches are general and illustrate how any
component of a signal transduction pathway or transcription factor
could be evolved using RSR and the screens and selections described
above. For example, these specific methods could be used to evolve
7-TM receptors with specificity for novel ligands, specificity of
nuclear receptors for novel ligands (for example to obtain
herbicide or other small molecule-inducible expression of genes of
interest in transgenic plants, such that a given set of genes can
be induced upon treatment with a given chemical agent, specificity
of transcription factors to be responsive to viral factors (thus
inducing antiviral or lethal genes in cells expressing this
transcription factor [transgenics or cells treated with gene
therapy constructs]), or specificity of transcription factors for
activity in cancer cells (for example p53 deficient cells, thus
allowing one to infect with gene therapy constructs expressing
conditionally lethal genes in a tumor specific fashion).
[0359] The following examples are offered by way of illustration,
not by way of limitation.
EXPERIMENTAL EXAMPLES
[0360] I. Evolution of BIAP
[0361] A preferred strategy to evolve BIAP is as follows. A codon
usage libary is constructed from 60-mer oligonucleotides such that
the central 20 bases of each oligo specifies the wild type protein,
but encodes the wild-type protein sequence with degenerate codons.
Preferably, very rare codons for the prokaryotic host of choice,
such as E. coli, are not used. The 20 bases at each end of the
oligo use non-degenerate, but preferred, codons in E. coli. The
oligonucleotides are assembled into full-length genes as described
above. The assembled products are cloned into an expression vector
by techniques well known in the art. In some embodiments, the codon
usage library is expressed with a library of secretory leader
sequences, each of which directs the encoded BIAP protein to the E.
coli periplasm. A library of leader sequences is used to optimize
the combination of leader sequence and mutant. Examples of leader
sequences are reviewed by Schatz et al. (Ann Rev. Genet. 24:215-248
(1990)). The cloned BIAP genes are expressed under the control of
an inducible promoter such as the arabinose promoter.
Arabinose-induced colonies are screened by spraying with a
substrate for BIAP, bromo-chloro-indolyl phosphate (BCIP). The
bluest colonies are picked visually and subjected to the RSR
procedures described herein.
[0362] The oligonucleotides for contruction of the codon usage
library are listed in Table II. The corresponding locations of
these promoters is provide in FIG. 1.
2TABLE II 1. AACCCTCCAG TTCCGAACCC CATATGATGA TCACCCTGCG TAAACTGCCG
2. AACCCTCCAG TTCCGAACCC CATATGAAAA AAACCGCT 3. AACCCTCCAG
TTCCGAACCC ATATACATAT GCGTGCTAAA 4. AACCCTCCAG TTCCGAACCC
CATATGAAAT ACCTGCTGCC GACC 5. AACCCTCCAG TTCCGAACCC GATATACATA
TGAAACAGTC 6. TGGTGTTATG TCTGCTCAGG CDATGGCDGT DGAYTTYCAY
CTGGTTCCGG TTGAAGAGGA 7. GGCTGGTTTC GCTACCGTTG CDCARGCDGC
DCCDAARGAY CTGGTTCCGG TTGAAGAGGA 8. CACCCCGATC GCTATCTCTT
CYTTYGCDTC YACYGGYTCY CTGGTTCCGG TTGAAGAGGA 9. GCTGCTGGCT
GCTCAGCCGG CDATGGCDAT GGAYATYGGY CTGGTTCCGG TTGAAGAGGA 10.
TGCCGCTGCT GTTCACCCCG GTDACYAARG CDGCDCARGT DCTGGTTCCG GTTGAAGAGG A
11. CCCGGCTTTC TGGAACCGTC ARGCDGCDCA RGCDCTGGAC GTTGCTAAAA
AACTGCAGCC 12. ACGTTATCCT GTTCCTGGGT GAYGGYATGG GYGTDCCDAC
CGTTACCGCT ACCCGTATCC 13. AAACTGGGTC CGGAAACCCC DCTGGCDATG
GAYCARTTYC CGTACGTTGC TCTGTCTAAA 14. GGTTCCGGAC TCTGCTGGTA
CYGCDACYGC DTAYCTGTGC GGTGTTAAAG GTAACTACCG 15. CTGCTCGTTA
CAACCAGTGC AARACYACYC GYGGYAAYGA AGTTACCTCT GTTATGAACC 16.
TCTGTTGGTG TTGTTACCAC YACYCGYGTD CARCAYGCDT CTCCGGCTGG TGCTTACGCT
17. GTACTCTGAC GCTGACCTGC CDGCDGAYGC DCARATGAAC GGTTGCCAGG
ACATCGCTGC 18. ACATCGACGT TATCCTGGGT GGYGGYCGYA ARTAYATGTT
CCCGGTTGGT ACCCCGGACC 19. TCTGTTAACG GTGTTCGTAA RCGYAARCAR
AAYCTGGTDC AGGCTTGGCA GGCTAAACAC 20. GAACCGTACC GCTCTGCTGC
ARGCDGCDGA YGAYTCYTCT GTTACCCACC TGATGGGTCT 21. AATACAACGT
TCAGCAGGAC CAYACYAARG AYCCDACYCT GCAGGAAATG ACCGAAGTTG 22.
AACCCGCGTG GTTTCTACCT GTTYGTDGAR GGYGGYCGYA TCGACCACGG TCACCACGAC
23. GACCGAAGCT GGTATGTTCG AYAAYGCDAT YGCDAARGCT AACGAACTGA
CCTCTGAACT 24. CCGCTGACCA CTCTCACGTT TTYTCYTTYG GYGGYTAYAC
CCTGCGTGGT ACCTCTATCT 25. GCTCTGGACT CTAAATCTTA YACYTCYATY
CTGTAYGGYA ACGGTCCGGG TTACGCTCTG 26. CGTTAACGAC TCTACCTCTG
ARGAYCCDTC YTAYCARCAG CAGGCTGCTG TTCCGCAGGC 27. AAGACGTTGC
TGTTTTCGCT CGYGGYCCDC ARGCDCAYCT GGTTCACGGT GTTGAAGAAG 28.
ATGGCTTTCG CTGGTTGCGT DGARCCDTAY ACYGAYTGYA ACCTGCCGGC TCCGACCACC
29. TGCTCACCTG GCTGCTTMAC CDCCDCCDCT GGCDCTGCTG GCTGGTGCTA
TGCTGCTCCT C 30. TTCCGCCTCT AGAGAATTCT TARTACAGRG THGGHGCCAG
GAGGAGCAGC ATAGCACCAG CC 31. AAGCAGCCAG GTGAGCAGCG TCHGGRATRG
ARGTHGCGGT GGTCGGAGCC GGCAGGTT 32. CGCAACCAGC GAAAGCCATG ATRTGHGCHA
CRAARGTYTC TTCTTCAACA CCGTGAACCA 33. GCGAAAACAG CAACGTCTTC
RCCRCCRTGR GTYTCRGAHG CCTGCGGAAC AGCAGCCTGC 34. AGAGGTAGAG
TCGTTAACGT CHGGRCGRGA RCCRCCRCCC AGAGCGTAAC CCGGACCGTT 35.
AAGATTTAGA GTCCAGAGCT TTRGAHGGHG CCAGRCCRAA GATAGAGGTA CCACGCAGGG
36. ACGTGAGAGT GGTCAGCGGT HACCAGRATC AGRGTRTCCA GTTCAGAGGT
CAGTTCGTTA 37. GAACATACCA GCTTCGGTCA GHGCCATRTA HGCYTTRTCG
TCGTGGTGAC CGTGGTCGAT 38. GGTAGAAACC ACGCGGGTTA CGRGAHACHA
CRCGCAGHGC AACTTCGGTC ATTTCCTGCA 39. TCCTGCTGAA CGTTGTATTT
CATRTCHGCH GGYTCRAACA GACCCATCAG GTGGGTAACA 40. CAGCAGAGCG
GTACGGTTCC AHACRTAYTG HGCRCCYTGG TGTTTAGCCT GCCAAGCCTG 41.
TACGAACACC GTTAACAGAA GCRTCRTCHG GRTAYTCHGG GTCCGGGGTA CCAACCGGGA
42. CCCAGGATAA CGTCGATGTC CATRTTRTTH ACCAGYTGHG CAGCGATGTC
CTGGCAACCG 43. CAGGTCAGCG TCAGAGTACC ARTTRCGRTT HACRGTRTGA
GCGTAAGCAC CAGCCGGAGA 44. TGGTAACAAC ACCAACAGAT TTRCCHGCYT
TYTTHGCRCG GTTCATAACA GAGGTAACTT 45. CACTGGTTGT AACGAGCAGC
HGCRGAHACR CCRATRGTRC GGTAGTTACC TTTAACACCG 46. ACCAGCAGAG
TCCGGAACCT GRCGRTCHAC RTTRTARGTT TTAGACAGAG CAACGTACGG 47.
GGGTTTCCGG ACCCAGTTTA CCRTTCATYT GRCCYTTCAG GATACGGGTA GCGGTAACGG
48. CCCAGGAACA GGATAACGTT YTTHGCHGCR GTYTGRATHG GCTGCAGTTT
TTTAGCAACG 49. ACGGTTCCAG AAAGCCGGGT CTTCCTCTTC AACCGGAACC AG 50.
CCTGAGCAGA CATAACACCA GCHGCHACHG CHACHGCCAG CGGCAGTTTA CGCAGGGTGA
51. ACCGGGGTGA ACAGCAGCGG CAGCAGHGCC AGHGCRATRG TRGACTGTTT
CATATGTATA TC 52. GCCGGCTGAG CAGCCAGCAG CAGCAGRCCH GCHGCHGCGG
TCGGCAGCAG GTAGTTTCA 53. AAGAGATAGC GATCGGGGTG GTCAGHACRA
TRCCCAGCAG TTTAGCACGC ATATGTATAT 54. CAACGGTAGC GAAACCAGCC
AGHGCHACHG CRATHGCRAT AGCGGTTTTT TTCATATG 55 AGAATTCTCT AGAGGCGGAA
ACTCTCCAAC TCCCAGGTT 56. TGAGAGGTTG AGGGTCCAAT TGGGAGGTCA AGGCTTGGG
All oligonucleotides listed 5' to 3'. The code for degenerate
positions is: R: A or G; Y: C or T; H: A or C or T; D: A or G or
T.
[0363] II. Mammalian Surface Display
[0364] During an immune response antibodies naturally undergo a
process of affinity maturation resulting in mutant antibodies with
improved affinities for their cognate antigens. This process is
driven by somatic hypermutation of antibody genes coupled with
clonal selection (Berek and Milstein, Immun. Rev. 96:23-41 (1987)).
Patten et al. (Science 271:1086-1091 (1996)) have reconstructed the
progression of a catalytic antibody from the germline sequence,
which binds a p-nitrophenylphosphonate hapten with an affinity of
135 micromolar, to the affinity matured sequence which has acquired
nine somatic mutations and binds with an affinity of 10 nanomolar.
The affinity maturation of this antibody can be recapitulated and
improved upon using cassette mutagenesis of the CDR's (or random
mutagenesis such as with PCR), mammalian display, FACS selection
for improved binding, and RSR to rapidly evolve improved affinity
by recombining mutations encoding improved binding.
[0365] Genomic antibody expression shuttle vectors similar to those
described by Gascoigne et al. (Proc. Natl. Acad. Sci. (U.S.A.)
84:2936-2940 (1987)) are constructed such that libraries of mutant
V region exons can be readily cloned into the shuttle vectors. The
kappa construct is cloned onto a plasmid encoding puromycin
resistance and the heavy chain is cloned onto a neomycin resistance
encoding vector. The cDNA derived variable region sequences
encoding the mature and germline heavy and light chain V regions
are reconfigured by PCR mutagenesis into genomic exons flanked by
Sfi I sites with complementary Sfi I sites placed at the
appropriate locations in the genomic shuttle vectors. The
oligonucleotides used to create the intronic Sfi I sites flanking
the VDJ exon are: 5' Sfi I: 5'-TTCCATTTCA TACATGGCCG AAGGGGCCGT
GCCATGAGGA TTTT-3'; 3' Sfi I: 5'-TTCTAAATG CATGTTGGCC TCCTTGGCCG
GATTCTGAGC CTTCAGGACC A-3'. Standard PCR mutagenesis protocols are
applied to produce libraries of mutants wherein the following sets
of residues (numbered according to Kabat, Sequences of Proteins of
Immunological Interest, U.S. Dept of Health and Human Services,
1991) are randomized to NNK codons (GATC,GATC,GC):
3 Chain CDR Mutated residues V-L 1 30, 31, 34 V-L 2 52, 53, 55 V-H
2 55, 50, 05 V-H "4" 74, 70, 78
[0366] Stable transfectant lines are made for each of the two light
and heavy chain constructs (mature and germline) using the B cell
myeloma AG8-653 (a gift from J. Kearney) as a host using standard
electroporation protocols. Libraries of mutant plasmids encoding
the indicated libraries of V-L mutants are transfected into the
stable transformant expressing the germline V-H; and the V-H
mutants are transfected into the germline V-L stable transfectant
line. In both cases, the libraries are introduced by protoplast
fusion (Sambrook et al., Molecular Cloning, CSH Press (1987)) to
ensure that the majority of transfected cells receive one and only
one mutant plasmid sequence (which would not be the case for
electroporation where the majority of the transfected cells would
receive many plasmids, each expressing a different mutant
sequence).
[0367] The p-nitrophenylphosphonate hapten (JWJ-1) recognized by
this antibody is synthesized as described by Patten et al. (Science
271:1086-1091 (1996)) JWJ-1 is coupled directly to
5-(((2-aminoethyl)thio)acetyl)fluorescein (Molecular Probes, Inc.,
by formation of an amide bond using a standard coupling chemistry
such as EDAC (March, Advanced Organic Chemistry, Third edition,
John Wiley and Sons, 1985) to give a monomeric JWJ-1-FITC probe. A
"dimeric" conjugate (two molecules of JWJ-1 coupled to a FACS
marker) is made in order to get a higher avidity probe, thus making
low affinity interactions (such as with the germline antibody) more
readily detected by FACS. This is generated by staining with Texas
Red conjugated o an anti-fluorescein antibody in the presence of
two equivalents of JWJ-1-FITC. The bivalent structure of IgG then
provides a homogeneous bivalent reagent. A spin column is used to
remove excess JWJ-1-FITC molecules that are not bound to the
anti-FITC reagent. A tetravalent reagent is made as follows. One
equivalent of biotin is coupled with EDAC to two equivalents of
ethylenediamine, and this is then be coupled to the free
carboxylate on JWJ-1. The biotiylated JWJ-1 product is purified by
ion exchange chromatography and characterized by mass spectrometry.
FITC labelled avidin is incubated with the biotinylated JWJ-1 in
order to generate a tetravalent probe.
[0368] The FACS selection is performed as follows, according to a
protocol similar to that of Panka et al. (Proc. Natl. Acad. Sci.
(U.S.A.) 85:3080-3084 (1988)). After transfection of libraries or
mutant antibody genes by the method of protoplast fusion (with
recovery for 36-72 hours), the cells are incubated on ice with
fluorescently labelled hapten. The incubation is done on ice to
minimize pinocytosis of the FITC conjugate which may contribute to
nonspecific background. The cells are then sorted on the FACS
either with or without a washing step. FACSing without a washing
step is preferable because the off rate for the germline antibody
prior to affinity maturation is expected to be very fast (>0.1
sec-1; Patten et al., Science 271:1086-1091 (1996)); a washing step
adds a complicating variable. The brightest 0.1-10% of the cells
are collected.
[0369] Four parameters are manipulated to optimize the selection
for increased binding: monomeric vs dimeric vs tetrameric hapten,
concentration of hapten used in the staining reaction (low
concentration selects for high affinity Kd's ), time between
washing and FACS (longer time selects for low off rates), and
selectivity in the gating (i.e. take the top 0.1% to 10%, move
preferably the top 0.1%). The constructs expressing the germline,
mature, and both combinations of half germline are used as controls
to optimize this selectivity.
[0370] Plasmids are recovered from the FACS selected cells by the
transformation of an E. coli host with Hirt supernatants.
Alternatively, the mutant V gene exons are PCR-amplified from the
FACS selected cells. The recovered V gene exons are subjected to
RSR, recloned into the corresponding genomic shuttle vector, and
the procedure recursively applied until the mean fluorescence
intensity has increased. A relevant positive control for improved
binding is transfection with the affinity matured 48G7 exons
(Patten et al., op. cit.).
[0371] In a further experiment, equal numbers of germline and each
of the two half germline transfectants are mixed. The brightest
cells are selected under conditions described above. The V genes
are recovered by PCR, recloned into expression vectors, and
co-transfected, either two plasmids per E. coli followed by
protoplast fusion, or by bulk electroporation. The mean fluorescent
intensity of the transfectants should increase due to enrichment of
mature relative to germline V regions.
[0372] This methodology can be applied to evolve any
receptor-ligand or binding partner interaction. Natural expression
formats can be used to express libraries of mutants of any receptor
for which one wants to improve the affinity for the natural or
novel ligands. Typical examples would be improvement of the
affinity of T cell receptors for ligands of interest (i.e.
MHC/tumor peptide antigen complexes) or TNF receptor for TNF
(soluble forms of TNF receptors are used therapeutically to
neutralize TNF activity).
[0373] This format can also be used to select for mutant forms of
ligands by expressing the ligand in a membrane bound form with an
engineered membrane anchor by a strategy analogous to that of
Wettstein et al. (J. Exp. Med. 174:219-28 (1991)). FACS selection
is then performed with fluorescently labelled receptor. In this
format one could, for example, evolve improved receptor antagonists
from naturally occurring receptor antagonists (IL1 receptor
antagonist, for example). Mutant forms of agonists with improved
affinity for their cognate receptors could also be evolved in this
format. These mutants would be candidates for improved agonists or
potent receptor antagonists, analogous to reported antagonistic
mutant forms of IL3.
[0374] III. Evolution of Aloha Interferon
[0375] There are at hand 18 known non-allelic human
interferon-alpha (INF-.alpha.) genes, with highly related primary
structures 78-95% identical) and with a broad range of biological
activities. Many hybrid interferons with interesting biological
activities differing from the parental molecules have been
described (reviewed by Horisberger and Di Marco, Pharm. Ther.
66:507-534 (1995)). A consensus human alpha interferon, IFN-Con1,
has been constructed synthetically wherein the most common residue
in fourteen known IFN-.alpha.'s has been put at each position, and
it compares favorably with the naturally occurring interferons
(Ozes et al., J. Interferon Res. 12:55-59 (1992)). This IFN
contains 20 amino acid changes relative to IFN-.alpha.2a, the
INF-.alpha. to which it is most closely related. IFN-Con1 has
10-fold higher specific antiviral activity than any known natural
IFN subtype. IFN-.alpha. Con1 has in vitro activities 10 to 20 fold
higher than that of recombinant IFN .alpha.-2a (the major IFN used
clinically) in antiviral, antiproliferative and NK cell activation.
Thus, there is considerable interest in producing interferon
hybrids which combine the most desirable traits from two or more
interferons. However, given the enormous number of potential
hybrids and the lack of a crystal structure of IFN -.alpha. or of
the IFN-.alpha. receptor, there is a perceived impasse in the
development of novel hybrids (Horisberger and Di Marco, Pharm.
Ther. 66:507-534 (1995)).
[0376] The biological effects of IFN-.alpha.'s are diverse, and
include such properties as induction of antiviral state (induction
of factors that arrest translation and degrade mRNA); inhibition of
cell growth; induction of Class I and Class II MHC; activation of
monocytes and macrophages; activation of natural killer cells;
activation of cytotoxic T cells; modulation of Ig synthesis in B
cells; and pyrogenic activity.
[0377] The various IFN-.alpha.'s subtypes have unique spectra of
activities on different target cells and unique side effect
profiles (Ortaldo et al., Proc. Natl. Acad. Sci. (U.S.A.)
81:4926-4929 (1984); Overall et al., J. Interferon Res. 12:281-288
(1992); Fish and Stebbing, Biochem. Biophys. Res. Comm. 112:537-546
(1983); Weck et al., J. Gen. Virol. 57:233-237 (1981)). For
example, human IFN.alpha. has very mild side effects but low
antiviral activity. Human IFN.alpha.8 has very high antiviral
activity, but relatively severe side effects. Human IFN.alpha.7
lacks NK activity and blocks NK stimulation by other INF.alpha.'s.
Human IFN-.alpha. J lacks the ability to stimulate NK cells, but it
can bind to the IFN-.alpha. receptor on NK cells and block the
stimulatory activity of IFN-.alpha.A (Langer et al., J. Interferon
Res. 6:97-105 (986)).
[0378] The therapeutic applications of interferons are limited by
diverse and severe side effect profiles which include flu-like
symptoms, fatigue, neurological disorders including hallucination,
fever, hepatic enzyme elevation, and leukopenia. The multiplicity
of effects of IFN-.alpha.'s has stimulated the hypothesis that
there may be more than one receptor or a multicomponent receptor
for the IFN-.alpha. family (R. Hu et al., J. Biol. Chem.
268:12591-12595 (1993)). Thus, the existence of abundant naturally
occurring diversity within the human alpha IFN's (and hence a large
sequence space of recombinants) along with the complexity of the
IFN-.alpha. receptors and activities creates an opportunity for the
construction of superior hybrids.
[0379] A. Complexity of the Sequence Space
[0380] FIG. 2 shows the protein sequences of 11 human IFN-.alpha.'s
. The differences from consensus are indicated. Those positions
where a degenerate codon can capture all of the diversity are
indicated with an asterisk. Examination of the aligned sequences
reveals that there are 57 positions with two, 15 positions with
three, and 4 positions with four possible amino acids encoded in
this group of alpha interferon genes. Thus, the potential diversity
encoded by permutation of all of this naturally occurring diversity
is: 2.sup.57.times.3.sup.15.times.4.sup.4=5- .3.times.10.sup.26.
Among these hybrids, of the 76 polymorphisms spread over a total of
175 sites in the 11 interferon genes, 171 of the 175 changes can be
incorporated into homologue libraries using single degenerate
codons at the corresponding positions. For example, Arg, Trp and
Gly can all be encoded by the degenerate codon [A,T,G]GG. Using
such a strategy, 1.3.times.10.sup.25 hybrids can be captured with a
single set of degenerate oligonucleotides. As is evident from
Tables III to VI, 27 oligonucleotides is sufficient to shuffle all
eleven human alpha interferons. Virtually all of the natural
diversity is thereby encoded and fully permuted due to degeneracies
an the nine "block" oligonucleotides in Table V.
[0381] B. Properties of a "Coarse Grain" Search of Homologue
Sequence Space
[0382] The modelled structure of IFN alpha (Kontsek, Acta Vir.
38:345-360 (1994)) has been divided into nine segments based on a
combination of criteria of maintaining secondary structure elements
as single units and placing/choosing placement of the segment
boundaries in regions of high identity. Hence, one can capture the
whole family with a single set of mildly degenerate
oligonucleotides. Table III and FIG. 2 give the precise locations
of these boundaries at the protein and DNA levels respectively. It
should be emphasized that this particular segmentation scheme is
arbitrary and that other segmentation schemes could also be
pursued. The general strategy does not depend on placement of
recombination boundaries at regions of high identity between the
family members or on any particular algorithm for breaking the
structure into segments.
4TABLE III Segmentation Scheme for Alpha Interferon # Permutations
of all Segment Amino Acids # Alleles Sequence Variations 1 1-21 5
1024 2 22-51 10 6.2 .times. 10.sup.4 3 52-67 6 96 4 68-80 7 1024 5
81-92 7 192 6 93-115 10 2.5 .times. 10.sup.5 7 116-131 4 8 8
132-138 4 8 9 139-167 9 9216
[0383] Many of the IFN's are identical over some of the segments,
and thus there are less than eleven different "alleles" of each
segment. Thus, a library consisting of the permutations of the
segment "alleles" would have a potential complexity of
2.1.times.10.sup.7 (5 segment #1's times 10 segment #2's.times. . .
. .times.9 segment #9's ). This is far more than can be examined in
most of the screening procedures described, and thus this is a good
problem for using RSR to search the sequence space.
[0384] C. Detailed Strategies for Using RSR to Search the IFN-alpha
Homologue Sequence Space
[0385] The methods described herein for oligo directed shuffling
(i.e. bridge oligonucleotides) are employed to construct libraries
of interferon alpha hybrids, and the general methods described
above are employed to screen or select these mutants for improved
function. As there are numerous formats in which to screen or
select for improved interferon activity, many of which depend on
the unique properties of interferons, exemplary descriptions of IFN
based assays are described below.
[0386] D. A Protocol for a Coarse Grain Search of Hybrid IFN Alpha
Sequence Space
[0387] In brief, libraries are constructed wherein the 11
homologous forms of the nine segments are permuted (note that in
many cases two homologues are identical over a given segment). All
nine segments are PCR-amplified out of all eleven IFN alpha genes
with the eighteen oligonucleotides listed in Table IV, and
reassembled into full length genes with oligo directed
recombination. An arbitrary number, e.g., 1000, clones from the
library are prepared in a 96-well expression/purification format.
Hybrids with the most potent antiviral activities are screened.
Nucleic acid is recovered by PCR amplification, and subjected to
recombination using bridge oligonucleotides. These steps are
repeated until candidates with desired properties are obtained.
[0388] E. Strategies for Examining the Space of >10.sup.26 Fine
Grain Hybrids
[0389] In brief, each of the nine segments is synthesized with one
degenerate oligo per segment. Degeneracies are chosen to capture
all of the IFN-alpha diversity that can be captured with a single
degenerate codon without adding any non-natural sequence. A second
set of degenerate oligonucleotides encoding the nine segments is
generated wherein all of the natural diversity is captured, but
additional non-natural mutations are included at positions where
necessitated by the constraints fo the genetic code. In mose cases
all of the diversity can be captured with a single degenerate
codon; in some cases a degenerate codon will capture all of the
natural diversity but will add one non-natural mutation; at a few
positions it is not possible to capture the natural diversity
without putting in a highly degenerate codon which will create more
than one non-natural mutation. It is at these positions that this
second set of oligonucleotides will differ from the first set by
being more inclusive. Each of the nine synthetic segments is then
amplified by PCR with the 18 PCR oligonucleotides. Full length
genes using the oligo directed recombination method are generated,
transfected into a host, and assayed for hybrids with desired
properties. The best hybrids from (e.g, the top 10%, 1% or 0.1%;
preferably the top 1%) are subjected to RSR and the process
repeated until a candidate with the desired properties is
obtained.
[0390] F. "Non-gentle" Fine Grain Search
[0391] On the one hand, one could make libraries wherein each
segment is derived from the degenerate synthetic oligonucleotides
which will encode random permutations of the homologue diversity.
In this case, the initial library will very sparsely search the
space of >10.sup.25 possible fine grain hybrids that are
possible with this family of genes. One could proceed by breeding
positives together from this search. However, there would be a
large number of differences between independent members of such
libraries, and consequently the breeding process would not be very
"gentle" because pools of relatively divergent genes would be
recombined at each step.
[0392] G. "Gentle" Fine Grain Search
[0393] One way to make this approach more "gentle" would be obtain
a candidate starting point and to gently search from there. This
starting point could be either one of the natural
IFN-.alpha.alpha's (such as IFN alpha-2a which is the one that is
being used most widely therapeutically), the characterized IFN-Conl
consensus interferon, or a hit from screening the shuffled
IFN-alpha's described above. Given a starting point, one would make
separate libraries wherein one breeds the degenerate segment
libraries one at a time into the founder sequence. Improved hits
from each library would then be bred together to gently build up
mutations all throughout the molecule.
[0394] H. Functional Cellular Assays
[0395] The following assays, well known in the art, are used to
screen IFN alpha mutants, inhibition of viral killing; standard
error of 30-50%; inhibition of plaque forming units; very low
standard error (can measure small effects); reduced viral yield
(useful for nonlethal, nonplaque forming viruses); inhibition of
cell growth (3H-thymidine uptake assay; activation of NK cells to
kill tumor cells; suppression of tumor formation by human INF
administered to nude mice engrafted with human tumors (skin tumors
for example).
[0396] Most of these assays are amenable to high throughput
screening. Libraries of recombinant IFN alpha mutants are expressed
and purified in high throughput formats such as expression, lysis
and purification in a 96-well format using anti-IFN antibodies or
an epitope tag and affinity resin. The purified IFN preparations
are screened in a high throughput format, scored, and the mutants
encoding the highest activities of interest are subjected to
further mutagenesis, such as RSR, and the process repeated until a
desired level of activity is obtained.
[0397] I. Phage Display
[0398] Standard phage display formats are used to display
biologically active IFN. Libraries of chimeric IFN genes are
expressed in this format and are selected (positively or
negatively) for binding (or reduced binding) to one or more
purified IFN receptor preparations or to one or more IFN receptor
expressing cell types.
[0399] J. GFP or Luciferase Under Control of IFN-Alpha Dependent
Promoter
[0400] Protein expressed by mutants can be screened in high
throughput format on a reporter cell line which expresses GFP or
luciferase under the control of an IFN alpha responsive promoter,
such as an MHC Class I promoter driving GFP expression.
[0401] K. Stimulation of Target Cells with Intact Infections
Particles
[0402] Purification of active IFN will limit the throughput of the
assays described above. Expression of active IFN alpha on
filamentous phage M13 would allow one to obtain homogenous
preparations of IFN mutants in a format where thousands or tens of
thousands of mutants could readily be handled. Gram et al. (J. Imm.
Meth. 161:169-176 (1993)) have demonstrated that human IL3, a
cytokine with a protein fold similar in topology to IFN alpha, can
be expressed on the surface of M13 and that the resultant phage can
present active IL3 to IL3 dependent cell lines. Similarly, Saggio
et al. (Gene 152:35-39 (1995)) have shown that human ciliary
neurotrophic factor, a four helix bundle cytokine, is biologically
active when expressed on phage at concentrations similar to those
of the soluble cytokine. Analogously, libraries of IFN alpha
mutants on M13 can be expressed and lysates of defined titre used
to present biologically active IFN in the high throughput assays
and selections described herein.
[0403] The following calculation supports the feasibility of
applying this technology to IFN alpha. Assuming (1) titres of
1.times.10.sup.10 phage/ml with five active copies of interferon
displayed per phage, and (2) that the displayed interferon is
equivalently active to soluble recombinant interferon it may well
be more potent due to multi-valency, the question then is whether
one can reasonably expect to see biological activity.
(1.times.10.sup.10 phage/ml).times.(5 IFN molecules/phage).times.(1
mole/6.times.10.sup.23 molecules).times.(26,000
gm/mole).times.(10.sup.9 ng/gm)=2.2 ng/ml
[0404] The range of concentration used in biological assays is: 1
ng/ml for NK activation, 0.1-10 ng/ml for antiproliferative
activity on Eskol cells, and 0.1-1 ng/ml on Daudi cells (Ozes et
al., J. Interferon Res. 12:55-59 (1992)). Although some subtypes
are glycosylated, interferon alpha2a and consensus interferon are
expressed in active recombinant form in E. coli, so at least these
two do not require glycosylation for activity. Thus, IFN alpha
expressed on filamentous phage is likely to be biologically active
as phage lysates without further purification. Libraries of IFN
chimeras are expressed in phage display formats and scored in the
assays described above and below to identify mutants with improved
properties to be put into further rounds of RSR.
[0405] When one phage is sufficient to activate one cell due to the
high valency state of the displayed protein (five per phage in the
gene III format; hundreds per phage in the gene VIII format; tens
in the lambda gene V format), then a phage lysate can be used
directly at suitable dilution to stimulate cells with a GFP
reporter construct under the control of an IFN responsive promoter.
Assuming that the phage remain attached after stimulation,
expression and FACS purification of the responsive cells, one could
then directly FACS purify hybrids with improved activity from very
large libraries (up to and perhaps larger than 10.sup.7 phage per
FACS run).
[0406] A second way in which FACS is used to advantage in this
format is the following. Cells can be stimulated in a multiwell
format with one lysate per well and a GFP type reporter construct.
All stimulated cells are FACS purified to collect the brightest
cells, and the IFN genes recovered and subjected to RSR, with
iteration of the protocol until the desired level of improvement is
obtained. In this protocol the stimulation is performed with
individual concentrated lysates and hence the requirement that a
single phage be sufficient to stimulate the cell is relaxed.
Furthermore, one can gate to collect the brightest cells which, in
turn, should have the most potent phage attached to them.
[0407] L. Cell Surface Display Protocol for IFN Alpha Mutants
[0408] A sample protocol follows for the cell surface display of
IFN alpha mutants. This form of display has at least two advantages
over phage display. First, the protein is displayed by a eukaryotic
cell and hence can be expressed in a properly glycosylated form
which may be necessary for some IFN alphas (and other growth
factors). Secondly, it is a very high valency display format and is
preferred in detecting activity from very weakly active
mutants.
[0409] In brief, a library of mutant IFN's is constructed wherein a
polypeptide signal for addition of a phosphoinositol tail has been
fused to the carboxyl terminus, thus targeting the protein for
surface expression (Wettstein et al., J. Exp. Med. 174:219-28
(1991)). The library is used to transfect reporter cells described
above (luciferase reporter gene) in a microtiter format. Positives
are detected with a charge coupling device(CCD) camera. Nucleic
acids are recovered either by HIRT and retransformation of the host
or by PCR, and are subjected to RSR for further evolution.
[0410] M. Autocrine Display Protocol for Viral Resistance
[0411] A sample protocol follows for the autocrine display of IFN
alpha mutants. In brief, a library of IFN mutants is generated in a
vector which allows for induction of expression (i.e.
metallothionein promoter) and efficient secretion. The recipient
cell line carrying an IFN responsive reporter cassette [GFP or
luciferase] is induced by transfection with the mutant IFN
constructs. Mutants which stimulate the IFN responsive promoter are
detected by by FACS or CCD camera.
[0412] A variation on this format is to challenge transfectants
with virus and select for survivors. One could do multiple rounds
of viral challenge and outgrowth on each set of transfectants prior
to retrieving the genes. Multiple rounds of killing and outgrowth
allow an exponential amplification of a small advantage and hence
provide an advantage in detecting small improvements in viral
killing.
5TABLE IV Oligonucleotides needed for blockwise recombination: 18
Oligonucleotides for alpha interferon shuffling 1.
5'-TGT[G/A]ATCTG[C/T]CT[C/G]AGACC 2.
5'-GGCACAAATG[G/A/C]G[A/C]AGAATCTCTC 3. 5'-AGAGATTCT[G/T]C[C/T/-
G]CATTTGTGCC 4. 5'-CAGTTCCAGAAG[A/G]CT[G/C] [C/A]AGCCATC 5.
5'-GATGGCT[T/G] [G/C]AG[T/C]CTTCTGGAACTG 6.
5'-CTTCAATCTCTTCA[G/C]CACA 7. 5'-TGTG[G/C]TGAAGAGATTGAAG 8.
5'-GGA[T/A] [G/C]AGA[C/G] [C/G]CTCCTAGA 9. 5'-TCTAGGAG[G/C]
[G/C]TCT[G/C] [T/A]TCC 10. 5'-GAACTT[T/G/A] [T/A]CCAGCAA[A/C]TGAAT
11. 5'-ATTCA[T/G]TTGCTGG[A/T] [A/T/C]AAGTTC 12.
5'-GGACT[T/C]CATCCTGGCTGTG 13. 5'-CACAGCCAGGATG[G/A]AGTCC 14.
5'-AAGAATCACTCTTTATCT 15. 5'-AGATAAAGAGTGATTCTT 16.
5'-TGGGAGGTTGTCAGAGCAG 17. 5'-CTGCTCTGACAACCTCCCA 18.
5'-TCA[A/T]TCCTT[C/A]CTC[T/C]TTAA
[0413] Brackets indicate degeneracy with equal mixture of the
specified bases at those positions. The purpose of the degeneracy
is to allow this one set of primers to prime all members of the IFN
family with similar efficiency. The choice of the oligo driven
recombination points is important because they will get
"overwritten" in each cycle of breeding and hence cannot coevolve
with the rest of the sequence over many cycles of selection.
6TABLE V Oliaonucleotides needed for "fine grain" recombination of
natural diversity over each of the nine blocks Block # Length of
oligo required 1 76 2 95 3 65 4 56 5 51 6 93 7 50 8 62 9 80
[0414]
7TABLE VI Amino acids that can be reached by a single step mutation
in the codon of interest. Wild-Type Amino Amino acids reachable by
one Acid mutation W C, R, G, L Y F, S, C, H, N, D F L, T, V, S, Y,
C L S, W, F, I, M, V, P V F, L, I, M, A, D, E, G I F, L, M, V, T,
N, K, S, R A S, P, T, V, D, E, G G V, A, D, E, R, S, C, W M L, I,
V, T, K, R S F, L, Y, C, W, P, T, A, P, G, N, T, I T S, P, A, I, M,
N, K, S, R P S, T, A, L, H, Q, R C F, S, Y, R, G, N Y, H, K, D, S,
T, I Q Y, H, K, E, L, P, R H Y, Q, N, D, L, P, R D Y, H, N, E, V,
A, G E Q, K, D, V, A, G R L, P, H, Q, C, W, S, G, K, T, I, M K Q,
N, E, R, T, I, M
[0415] Based on this Table, the polymorphic positions in IFN alpha
where all of the diversity can be captured by a degenerate codon
have been identified. Oligonucleotides of the length indicated in
Table V above with the degeneracies inferred from Table VI are
synthesized.
[0416] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
[0417] All references cited herein are expressly incorporated in
their entirety for all purposes.
Sequence CWU 1
1
101 1 50 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 1
aaccctccag ttccgaaccc catatgatga tcaccctgcg taaactgccg 50 2 38 DNA
Artificial Sequence Description of Artificial Sequence degenerate
oligonucleotide used for codon usage library 2 aaccctccag
ttccgaaccc catatgaaaa aaaccgct 38 3 40 DNA Artificial Sequence
Description of Artificial Sequence degenerate oligonucleotide used
for codon usage library 3 aaccctccag ttccgaaccc atatacatat
gcgtgctaaa 40 4 44 DNA Artificial Sequence Description of
Artificial Sequence degenerate oligonucleotide used for codon usage
library 4 aaccctccag ttccgaaccc catatgaaat acctgctgcc gacc 44 5 40
DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 5
aaccctccag ttccgaaccc gatatacata tgaaacagtc 40 6 60 DNA Artificial
Sequence Description of Artificial Sequence degenerate
oligonucleotide used for codon usage library 6 tggtgttatg
tctgctcagg cdatggcdgt dgayttycay ctggttccgg ttgaagagga 60 7 60 DNA
Artificial Sequence Description of Artificial Sequence degenerate
oligonucleotide used for codon usage library 7 ggctggtttc
gctaccgttg cdcargcdgc dccdaargay ctggttccgg ttgaagagga 60 8 60 DNA
Artificial Sequence Description of Artificial Sequence degenerate
oligonucleotide used for codon usage library 8 caccccgatc
gctatctctt cyttygcdtc yacyggytcy ctggttccgg ttgaagagga 60 9 60 DNA
Artificial Sequence Description of Artificial Sequence degenerate
oligonucleotide used for codon usage library 9 gctgctggct
gctcagccgg cdatggcdat ggayatyggy ctggttccgg ttgaagagga 60 10 61 DNA
Artificial Sequence Description of Artificial Sequence degenerate
oligonucleotide used for codon usage library 10 tgccgctgct
gttcaccccg gtdacyaarg cdgcdcargt dctggttccg gttgaagagg 60 a 61 11
60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 11
cccggctttc tggaaccgtc argcdgcdca rgcdctggac gttgctaaaa aactgcagcc
60 12 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 12
acgttatcct gttcctgggt gayggyatgg gygtdccdac cgttaccgct acccgtatcc
60 13 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 13
aaactgggtc cggaaacccc dctggcdatg gaycarttyc cgtacgttgc tctgtctaaa
60 14 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 14
ggttccggac tctgctggta cygcdacygc dtayctgtgc ggtgttaaag gtaactaccg
60 15 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 15
ctgctcgtta caaccagtgc aaracyacyc gyggyaayga agttacctct gttatgaacc
60 16 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 16
tctgttggtg ttgttaccac yacycgygtd carcaygcdt ctccggctgg tgcttacgct
60 17 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 17
gtactctgac gctgacctgc cdgcdgaygc dcaratgaac ggttgccagg acatcgctgc
60 18 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 18
acatcgacgt tatcctgggt ggyggycgya artayatgtt cccggttggt accccggacc
60 19 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 19
tctgttaacg gtgttcgtaa rcgyaarcar aayctggtdc aggcttggca ggctaaacac
60 20 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 20
gaaccgtacc gctctgctgc argcdgcdga ygaytcytct gttacccacc tgatgggtct
60 21 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 21
aatacaacgt tcagcaggac cayacyaarg ayccdacyct gcaggaaatg accgaagttg
60 22 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 22
aacccgcgtg gtttctacct gttygtdgar ggyggycgya tcgaccacgg tcaccacgac
60 23 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 23
gaccgaagct ggtatgttcg ayaaygcdat ygcdaargct aacgaactga cctctgaact
60 24 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 24
ccgctgacca ctctcacgtt ttytcyttyg gyggytayac cctgcgtggt acctctatct
60 25 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 25
gctctggact ctaaatctta yacytcyaty ctgtayggya acggtccggg ttacgctctg
60 26 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 26
cgttaacgac tctacctctg argayccdtc ytaycarcag caggctgctg ttccgcaggc
60 27 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 27
aagacgttgc tgttttcgct cgyggyccdc argcdcayct ggttcacggt gttgaagaag
60 28 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 28
atggctttcg ctggttgcgt dgarccdtay acygaytgya acctgccggc tccgaccacc
60 29 61 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 29
tgctcacctg gctgcttmac cdccdccdct ggcdctgctg gctggtgcta tgctgctcct
60 c 61 30 62 DNA Artificial Sequence Description of Artificial
Sequence degenerate oligonucleotide used for codon usage library 30
ttccgcctct agagaattct tartacagrg thgghgccag gaggagcagc atagcaccag
60 cc 62 31 58 DNA Artificial Sequence Description of Artificial
Sequence degenerate oligonucleotide used for codon usage library 31
aagcagccag gtgagcagcg tchggratrg argthgcggt ggtcggagcc ggcaggtt 58
32 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 32
cgcaaccagc gaaagccatg atrtghgcha craargtytc ttcttcaaca ccgtgaacca
60 33 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 33
gcgaaaacag caacgtcttc rccrccrtgr gtytcrgahg cctgcggaac agcagcctgc
60 34 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 34
agaggtagag tcgttaacgt chggrcgrga rccrccrccc agagcgtaac ccggaccgtt
60 35 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 35
aagatttaga gtccagagct ttrgahgghg ccagrccraa gatagaggta ccacgcaggg
60 36 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 36
acgtgagagt ggtcagcggt haccagratc agrgtrtcca gttcagaggt cagttcgtta
60 37 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 37
gaacatacca gcttcggtca ghgccatrta hgcyttrtcg tcgtggtgac cgtggtcgat
60 38 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 38
ggtagaaacc acgcgggtta cgrgahacha crcgcaghgc aacttcggtc atttcctgca
60 39 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 39
tcctgctgaa cgttgtattt catrtchgch ggytcraaca gacccatcag gtgggtaaca
60 40 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 40
cagcagagcg gtacggttcc ahacrtaytg hgcrccytgg tgtttagcct gccaagcctg
60 41 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 41
tacgaacacc gttaacagaa gcrtcrtchg grtaytchgg gtccggggta ccaaccggga
60 42 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 42
cccaggataa cgtcgatgtc catrttrtth accagytghg cagcgatgtc ctggcaaccg
60 43 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 43
caggtcagcg tcagagtacc arttrcgrtt hacrgtrtga gcgtaagcac cagccggaga
60 44 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 44
tggtaacaac accaacagat ttrcchgcyt tytthgcrcg gttcataaca gaggtaactt
60 45 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 45
cactggttgt aacgagcagc hgcrgahacr ccratrgtrc ggtagttacc tttaacaccg
60 46 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 46
accagcagag tccggaacct grcgrtchac rttrtargtt ttagacagag caacgtacgg
60 47 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 47
gggtttccgg acccagttta ccrttcatyt grccyttcag gatacgggta gcggtaacgg
60 48 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 48
cccaggaaca ggataacgtt ytthgchgcr gtytgrathg gctgcagttt tttagcaacg
60 49 42 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 49
acggttccag aaagccgggt cttcctcttc aaccggaacc ag 42 50 60 DNA
Artificial Sequence Description of Artificial Sequence degenerate
oligonucleotide used for codon usage library 50 cctgagcaga
cataacacca gchgchachg chachgccag cggcagttta cgcagggtga 60 51 62 DNA
Artificial Sequence Description of Artificial Sequence degenerate
oligonucleotide used for codon usage library 51 accggggtga
acagcagcgg cagcaghgcc aghgcratrg trgactgttt catatgtata 60 tc 62 52
59 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 52
gccggctgag cagccagcag cagcagrcch gchgchgcgg tcggcagcag gtagtttca 59
53 60 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 53
aagagatagc gatcggggtg gtcaghacra trcccagcag tttagcacgc atatgtatat
60 54 58 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 54
caacggtagc gaaaccagcc aghgchachg crathgcrat agcggttttt ttcatatg 58
55 39 DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for codon usage library 55
agaattctct agaggcggaa actctccaac tcccaggtt 39 56 39 DNA Artificial
Sequence Description of Artificial Sequence degenerate
oligonucleotide used for codon usage library 56 tgagaggttg
agggtccaat tgggaggtca aggcttggg 39 57 18 DNA Artificial Sequence
Description of Artificial Sequence degenerate oligonucleotide used
for alpha interferon shuffling 57 tgtratctgy ctsagacc 18 58 23 DNA
Artificial Sequence Description of Artificial Sequence degenerate
oligonucleotide used for alpha interferon shuffling 58 ggcacaaatg
vgmagaatct ctc 23 59 22 DNA Artificial Sequence Description of
Artificial Sequence degenerate oligonucleotide used for alpha
interferon shuffling 59 agagattctk cbcatttgtg cc 22 60 24 DNA
Artificial Sequence Description of Artificial Sequence degenerate
oligonucleotide used for alpha interferon shuffling 60 cagttccaga
agrctsmagc catc 24 61 24 DNA Artificial Sequence Description of
Artificial Sequence degenerate oligonucleotide used for alpha
interferon shuffling 61 gatggctksa gycttctgga actg 24 62 19 DNA
Artificial Sequence Description of Artificial Sequence degenerate
oligonucleotide used for alpha interferon shuffling 62 cttcaatctc
ttcascaca 19 63 19 DNA Artificial Sequence Description of
Artificial Sequence degenerate oligonucleotide used for alpha
interferon shuffling 63 tgtgstgaag agattgaag 19 64 18 DNA
Artificial Sequence Description of Artificial Sequence degenerate
oligonucleotide used for alpha interferon shuffling 64 ggawsagass
ctcctaga 18 65 18 DNA Artificial Sequence Description of Artificial
Sequence degenerate oligonucleotide used for alpha interferon
shuffling 65 tctaggagss tctswtcc 18 66 21 DNA Artificial Sequence
Description of Artificial Sequence degenerate oligonucleotide used
for alpha interferon shuffling 66 gaacttdwcc agcaamtgaa t 21 67 21
DNA Artificial Sequence Description of Artificial Sequence
degenerate oligonucleotide used for alpha interferon shuffling 67
attcakttgc tggwhaagtt c 21 68 19 DNA Artificial Sequence
Description of Artificial Sequence degenerate oligonucleotide used
for alpha interferon shuffling 68 ggactycatc ctggctgtg 19 69 19 DNA
Artificial Sequence Description of Artificial Sequence degenerate
oligonucleotide used for alpha interferon shuffling 69 cacagccagg
atgragtcc 19 70 18 DNA Artificial Sequence Description of
Artificial Sequence degenerate oligonucleotide used for alpha
interferon shuffling 70 aagaatcact ctttatct 18 71 18 DNA Artificial
Sequence Description of Artificial Sequence degenerate
oligonucleotide used for alpha interferon shuffling 71 agataaagag
tgattctt 18 72 19 DNA Artificial Sequence Description of Artificial
Sequence degenerate oligonucleotide used for alpha interferon
shuffling 72 tgggaggttg tcagagcag 19 73 19 DNA Artificial Sequence
Description of Artificial Sequence degenerate oligonucleotide used
for alpha interferon shuffling 73 ctgctctgac aacctccca 19 74 18 DNA
Artificial Sequence Description of Artificial Sequence degenerate
oligonucleotide used for alpha interferon shuffling 74 tcawtccttm
ctcyttaa 18 75 166 PRT consensus alpha interferon 75 Cys Asp Leu
Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 1 5 10 15 Leu
Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25
30 Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe
35 40 45 Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln
Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala
Trp Glu Gln Ser 65 70 75 80 Leu Leu Glu Lys Phe Ser Thr Glu Leu Tyr
Gln Gln Leu Asn Asp Leu 85 90 95 Glu Ala Cys Val Ile Gln Glu Val
Gly Val Glu Glu Thr Pro Leu Met 100 105 110 Asn Glu Asp Ser Ile Leu
Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr
Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala
Glu Ile Met Arg Ser Leu Ser Phe Ser Thr Asn Leu Gln Lys 145 150 155
160 Arg Leu Arg Arg Lys Asp 165 76 166 PRT human alpha interferon
76 Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile
1 5 10 15 Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu
Lys Asp 20 25 30 Arg His Asp Phe Gly Leu Pro Gln Glu Glu Phe Asp
Gly Asn Gln Phe 35 40 45 Gln Lys Thr Gln Ala Ile Pro Val Leu His
Glu Met Ile Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser
Thr Glu Asp Ser Ser Ala Ala Trp Glu Gln Ser 65 70 75 80 Leu Leu Glu
Lys Phe Ser Thr Glu Leu Tyr Gln Gln Leu Asn Asn Leu 85 90 95 Glu
Ala Cys Val Ile Gln Glu Val Gly Met Glu Glu Thr Pro Leu Met 100 105
110 Asn Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr
115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu
Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Leu Ser Phe Ser Thr
Asn Leu Gln Lys 145 150 155 160 Arg Leu Arg Arg Lys Asp 165 77 166
PRT human alpha interferon 77 Cys Asp Leu Pro Gln Thr His Ser Leu
Gly Asn Arg Arg Ala Leu Ile 1 5 10 15 Leu Leu Ala Gln Met Gly Arg
Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30 Arg Pro Asp Phe Gly
Leu Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe 35 40 45 Gln Lys Thr
Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr 50 55 60 Phe
Asn Leu Phe Ser Thr Glu Asp Ser Ser Ala Ala Trp Glu Gln Ser 65 70
75 80 Leu Leu Glu Lys Phe Ser Thr Glu Leu Tyr Gln Gln Leu Asn Asn
Leu 85 90 95 Glu Ala Cys Val Ile Gln Glu Val Gly Met Glu Glu Thr
Pro Leu Met 100 105 110 Asn Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr
Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser
Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser
Leu Ser Phe Ser Thr Asn Leu Gln Lys 145 150 155 160 Ile Leu Arg Arg
Lys Asp 165 78 166 PRT human alpha interferon 78 Cys Asn Leu Ser
Gln Thr His Ser Leu Asn Asn Arg Arg Thr Leu Met 1 5 10 15 Leu Leu
Ala Gln Met Arg Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30
Arg His Asp Phe Glu Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe 35
40 45 Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Met Gln Gln
Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asn Ser Ser Ala Ala Trp
Asp Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln
Gln Met Asn Asp Leu 85 90 95 Glu Ala Cys Val Ile Gln Glu Val Gly
Val Glu Glu Thr Pro Leu Met 100 105 110 Asn Glu Asp Ser Ile Leu Ala
Val Lys Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Met Glu
Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu
Ile Met Arg Ser Leu Ser Phe Ser Thr Asn Leu Gln Lys 145 150 155 160
Arg Leu Arg Arg Lys Asp 165 79 166 PRT human alpha interferon 79
Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 1 5
10 15 Leu Leu Ala Gln Met Gly Arg Ile Ser His Phe Ser Cys Leu Lys
Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Glu Glu Glu Phe Asp Gly
His Gln Phe 35 40 45 Gln Lys Thr Gln Ala Ile Ser Val Leu His Glu
Met Ile Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Glu Asp Ser
Ser Ala Ala Trp Glu Gln Ser 65 70 75 80 Leu Leu Glu Lys Phe Ser Thr
Glu Leu Tyr Gln Gln Leu Asn Asp Leu 85 90 95 Glu Ala Cys Val Ile
Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met 100 105 110 Asn Val Asp
Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu
Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135
140 Arg Ala Glu Ile Met Arg Ser Leu Ser Phe Ser Thr Asn Leu Gln Lys
145 150 155 160 Arg Leu Arg Arg Lys Asp 165 80 166 PRT human alpha
interferon 80 Cys Asp Leu Pro Gln Thr His Ser Leu Gly His Arg Arg
Thr Met Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe
Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Arg Phe Pro Gln Glu
Glu Phe Asp Gly Asn Gln Phe 35 40 45 Gln Lys Ala Glu Ala Ile Ser
Val Leu His Glu Val Ile Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser
Thr Lys Asp Ser Ser Val Ala Trp Asp Glu Arg 65 70 75 80 Leu Leu Asp
Lys Leu Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu 85 90 95 Glu
Ala Cys Val Met Gln Glu Val Trp Val Gly Gly Thr Pro Leu Met 100 105
110 Asn Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr
115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu
Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Ser Ser Arg
Asn Leu Gln Glu 145 150 155 160 Arg Leu Arg Arg Lys Glu 165 81 166
PRT human alpha interferon 81 Cys Asp Leu Pro Gln Thr His Ser Leu
Arg Asn Arg Arg Ala Leu Ile 1 5 10 15 Leu Leu Ala Gln Met Gly Arg
Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Glu Phe Arg
Phe Pro Glu Glu Glu Phe Asp Gly His Gln Phe 35 40 45 Gln Lys Thr
Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr 50 55 60 Phe
Asn Leu Phe Ser Thr Glu Asp Ser Ser Ala Ala Trp Glu Gln Ser 65 70
75 80 Leu Leu Glu Lys Phe Ser Thr Glu Leu Tyr Gln Gln Leu Asn Asp
Leu 85 90 95 Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr
Pro Leu Met 100 105 110 Asn Glu Asp Phe Ile Leu Ala Val Arg Lys Tyr
Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Met Glu Lys Lys Tyr Ser
Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser
Phe Ser Phe Ser Thr Asn Leu Lys Lys 145 150 155 160 Gly Leu Arg Arg
Lys Asp 165 82 166 PRT human alpha interferon 82 Cys Asp Leu Pro
Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 1 5 10 15 Leu Leu
Ala Gln Met Arg Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30
Arg His Asp Phe Glu Phe Pro Gln Glu Glu Phe Asp Asp Lys Gln Phe 35
40 45 Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln
Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Leu
Asp Glu Thr 65 70 75 80 Leu Leu Asp Glu Phe Tyr Ile Glu Leu Asp Gln
Gln Leu Asn Asp Leu 85 90 95 Glu Ser Cys Val Met Gln Glu Val Gly
Val Ile Glu Ser Pro Leu Met 100 105 110 Tyr Glu Asp Ser Ile Leu Ala
Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu
Lys Lys Tyr Ser Ser Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu
Ile Met Arg Ser Phe Ser Leu Ser Ile Asn Leu Gln Lys 145 150 155 160
Arg Leu Lys Ser Lys Glu 165 83 166 PRT human alpha interferon 83
Cys Asp Leu Pro Glu Thr His Ser Leu Asp Asn Arg Arg Thr Leu Met 1 5
10 15 Leu Leu Ala Gln Met Ser Arg Ile Ser Pro Ser Ser Cys Leu Met
Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly
Asn Gln Phe 35 40 45 Gln Lys Ala Pro Ala Ile Ser Val Leu His Glu
Leu Ile Gln Gln Ile 50 55 60 Phe Asn Leu Phe Thr Thr Lys Asp Ser
Ser Ala Ala Trp Asp Glu Asp 65 70 75 80 Leu Leu Asp Lys Phe Cys Thr
Glu Leu Tyr Gln Gln Leu Asn Asp Leu 85 90 95 Glu Ala Cys Val Met
Gln Glu Glu Arg Val Gly Glu Thr Pro Leu Met 100 105 110 Asn Ala Asp
Ser Ile Leu Ala Val Lys Lys Tyr Phe Arg Arg Ile Thr 115 120 125 Leu
Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135
140 Arg Ala Glu Ile Met Arg Ser Leu Ser Leu Ser Thr Asn Leu Gln Glu
145 150 155 160 Arg Leu Arg Arg Lys Glu 165 84 166 PRT human alpha
interferon 84 Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg
Ala Leu Ile 1 5 10 15 Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe
Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Gln Glu
Glu Phe Asp Gly Asn Gln Phe 35 40 45 Gln Lys Ala Gln Ala Ile Ser
Val Leu His Glu Met Ile Gln Gln Thr 50 55 60 Phe Asn Leu Phe Ser
Thr Lys Asp Ser Ser Ala Ile Trp Glu Gln Ser 65 70 75 80 Leu Leu Glu
Lys Phe Ser Thr Glu Leu Asn Gln Gln Leu Asn Asp Met 85 90 95 Glu
Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met 100 105
110 Asn Val Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Gln Arg Ile Thr
115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu
Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Lys
Ile Phe Gln Glu 145 150 155 160 Arg Leu Arg Arg Lys Ser 165 85 166
PRT human alpha interferon 85 Cys Asp Leu Pro Gln Thr His Ser Leu
Gly Asn Arg Arg Ala Leu Ile 1 5 10 15 Leu Leu Ala Gln Met Gly Arg
Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30 Arg Pro Asp Phe Gly
Leu Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe 35 40 45 Gln Lys Thr
Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr 50 55 60 Phe
Asn Leu Phe Ser Thr Glu Asp Ser Ser Ala Ala Trp Glu Gln Ser 65 70
75 80 Leu Leu Glu Lys Phe Ser Thr Glu Leu Tyr Gln Gln Leu Asn Asn
Leu 85 90 95 Glu Ala Cys Val Ile Gln Glu Val Gly Met Glu Glu Thr
Pro Leu Met 100 105 110 Asn Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr
Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser
Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser
Leu Ser Phe Ser Thr Asn Leu Gln Lys 145 150 155 160 Ile Leu Arg Arg
Lys Asp 165 86 166 PRT human alpha interferon 86 Cys Asp Leu Pro
Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 1 5 10 15 Leu Leu
Ala Gln Met Gly Arg Ile Ser His Phe Ser Cys Leu Lys Asp 20 25 30
Arg Tyr Asp Phe Gly Phe Pro Gln Glu Val Phe Asp Gly Asn Gln Phe 35
40 45 Gln Lys Ala Gln Ala Ile Ser Ala Phe His Glu Met Ile Gln Gln
Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp
Asp Glu Thr 65 70 75 80 Leu Leu Asp Lys Phe Tyr Ile Glu Leu Phe Gln
Gln Leu Asn Asp Leu 85 90 95 Glu Ala Cys Val Thr Gln Glu Val Gly
Val Glu Glu Ile Ala Leu Met 100 105 110 Asn Glu Asp Ser Ile Leu Ala
Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Met Gly
Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu
Ile Met Arg Ser Phe Ser Phe Ser Thr Asn Leu Gln Lys 145 150 155 160
Gly Leu Arg Arg Lys Asp 165 87 501 DNA consensus alpha interferon
87 tgtgatctgc ctcagaccca cagcctgggt aataggaggg ccttgatact
cctggcacaa 60 atgggaagaa tctctccttt ctcctgcctg aaggacagac
atgactttgg atttccccag 120 gaggagtttg atggcaacca gttccagaag
gctcaagcca tctctgtcct ccatgagatg 180 atccagcaga ccttcaatct
cttcagcaca aaggactcat ctgctgcttg ggatgagagc 240 ctcctagaaa
aattttccac tgaactttac cagcaactga atgacctgga agcctgtgtg 300
atacaggagg ttggggtgga agagactccc ctgatgaatg aggactccat cctggctgtg
360 aggaaatact tccaaagaat cactctttat ctgacagaga agaaatacag
cccttgtgcc 420 tgggaggttg tcagagcaga aatcatgaga tccttctctt
tttcaacaaa cttgcaaaaa 480 agattaagga ggaaggattg a 501 88 501 DNA
human alpha interferon 88 tgtgatctgc ctcagaccca cagcctgggt
aataggaggg ccttgatact cctggcacaa 60 atgggaagaa tctctccttt
ctcctgcctg aaggacagac atgactttgg acttccccag 120 gaggagtttg
atggcaacca gttccagaag actcaagcca tccctgtcct ccatgagatg 180
atccagcaga ccttcaatct cttcagcaca gaggactcat ctgctgcttg ggaacagagc
240 ctcctagaaa aattttccac tgaactttac cagcaactga ataacctgga
agcatgtgtg 300 atagaggagg ttgggatgga agagactccc ctgatgaatg
aggactccat cctggctgtg 360 aggaaatact tccaaagaat cactctttat
ctaacagaga agaaatacag cccttgtgcc 420 tgggaggttg tcagagcaga
aatcatgaga tccctctctt tttcaacaaa cttgcaaaaa 480 agattaagga
ggaaggattg a 501 89 501 DNA human alpha interferon 89 tgtgatctgc
ctcagaccca cagcctgggt aataggaggg ccttgatact cctggcacaa 60
atgggaagaa tctctccttt ctcctgcctg aaggacagac ctgactttgg acttccccag
120 gaggagtttg atggcaacca gttccagaag actcaagcca tctctgtcct
ccatgagatg 180 atccagcaga ccttcaatct cttcagcaca gaggactcat
ctgctgcttg ggaacagagc 240 ctcctagaaa aattttccac tgaactttac
cagcaactga ataacctgga agcatgtgtg 300 atacaggagg ttgggatgga
agagactccc ctgatgaatg aggactccat cctggctgtg 360 aggaaatact
tccaaagaat cactctttat ctaacagaga agaaatacag cccttgtgcc 420
tgggaggttg tcagagcaga aatcatgaga tctctctctt tttcaacaaa cttgcaaaaa
480 atattaagga ggaaggattg a 501 90 501 DNA human alpha interferon
90 tgtaatctgt ctcaaaccca cagcctgaat aacaggagga ctttgatgct
catggcacaa 60 atgaggagaa tctctccttt ctcctgcctg aaggacagac
atgactttga atttccccag 120 gaggaatttg atggcaacca gttccagaaa
gctcaagcca tctctgtcct ccatgagatg 180 atgcagcaga ccttcaatct
cttcagcaca aagaactcat ctgctgcttg ggatgagacc 240 ctcctagaaa
aattctacat tgaacttttc cagcaaatga atgacctgga agcctgtgtg 300
atacaggagg ttggggtgga agagactccc ctgatgaatg aggactccat cctggctgtg
360 aagaaatact tccaaagaat cactctttat ctgatggaga agaaatacag
cccttgtgcc 420 tgggaggttg tcagagcaga aatcatgaga tccctctctt
tttcaacaaa cttgcaaaaa 480 agattaagga ggaaggattg a 501 91 501 DNA
human alpha interferon 91 tgtgatctgc ctcagaccca cagcctgggt
aataggaggg ccttgatact cctggcacaa 60 atgggaagaa tctctccttt
ctcatgcctg aaggacagac atgatttcgg attccccgag 120 gaggagtttg
atggccacca gttccagaag actcaagcca tctctgtcct ccatgagatg 180
atccagcaga ccttcaatct cttcagcaca gaggactcat ctgctgcttg ggaacagagc
240 ctcctagaaa aattttccac tgaactttac cagcaactga atgacctgga
agcatgtgtg 300 atacaggagg ttggggtgga agagactccc ctgatgaatg
tggactccat cctggctgtg 360 aggaaatact tccaaagaat cactctttat
ctaacagaga agaaatacag cccttgtgcc 420 tgggaggttg tcagagcaga
aatcatgaga tccctctcgt tttcaacaaa cttgcaaaaa 480 agattaagga
ggaaggattg a 501 92 501 DNA human alpha interferon 92 tgtgatctgc
ctcagaccca cagcctgggt cacaggagga ccatgatgct cctggcacaa 60
atgaggagaa tctctctttt ctcctgtctg aaggacagac atgacttcag atttccccag
120 gaggagtttg atggcaacca gttccagaag gctgaagcca tctctgtcct
ccatgaggtg 180 attcagcaga ccttcaatct cttcagcaca aaggactcat
ctgttgcttg ggatgagagg 240 cttctagaca aactctatac tgaactttac
cagcagctga atgacctgga agcctgtgtg 300 atgcaggagg tgtgggtggg
agggactccc ctgatgaatg aggactccat cctggctgtg 360 agaaaatact
tccaaagaat cactctctac ctgacagaga aaaagtacag cccttgtgcc 420
tgggaggttg tcagagcaga aatcatgaga tccttctctt catcaagaaa cttgcaagaa
480 aggttaagga ggaaggaata a 501 93 501 DNA human alpha interferon
93 tgtgatctgc ctcagaccca cagcctgcgt aataggaggg ccttgatact
cctggcacaa 60 atgggaagaa tctctccttt ctcctgcttg aaggacagac
atgaattcag attcccagag 120 gaggagtttg atggccacca gttccagaag
actcaagcca tctctgtcct ccatgagatg 180 atccagcaga ccttcaatct
cttcagcaca gaggactcat ctgctgcttg ggaacagagc 240 ctcctagaaa
aattttccac tgaactttac cagcaactga atgacctgga agcatgtgtg 300
atacaggagg ttggggtgga agagactccc ctgatgaatg aggactccat cctggctgtg
360 aggaaatact tccaaagaat cactctttat ctaatggaga agaaatacag
cccttgtgcc 420 tgggaggttg tcagagcaga aatcatgaga tccttctctt
tttcaacaaa
cttgaaaaaa 480 ggattaagga ggaaggattg a 501 94 501 DNA human alpha
interferon 94 tgtgatctgc ctcagactca cagcctgggt aacaggaggg
ccttgatact cctggcacaa 60 atgcgaagaa tctctccttt ctcctgcctg
aaggacagac atgactttga attcccccag 120 gaggagtttg atgataaaca
gttccagaag gctcaagcca tctctgtcct ccatgagatg 180 atccagcaga
ccttcaacct cttcagcaca aaggactcat ctgctgcttt ggatgagacc 240
cttctagatg aattctacat cgaacttgac cagcagctga atgacctgga gtcctgtgtg
300 atgcaggaag tgggggtgat agagtctccc ctgatgaatg aggacttcat
cctggctgtg 360 aggaaatact tccaaagaat cactctatat ctgacagaga
agaaatacag ctcttgtgcc 420 tgggaggttg tcagagcaga aatcatgaga
tccttctctt tatcaatcaa cttgcaaaaa 480 agattgaaga gtaaggaatg a 501 95
501 DNA human alpha interferon 95 tgtgatctcc ctgagaccca cagcctggat
aacaggagga ccttgatgct cctggcacaa 60 atgagcagaa tctctccttc
ctcctgtctg atggacagac atgactttgg atttccccag 120 gaggagtttg
atggcaacca gttccagaag gctccagcca tctctgtcct ccatgagctg 180
atccagcaga tcttcaacct cttctccaca aaagattcat ctgctgcttg ggatgaggac
240 ctcctagaca aattctgcac cgaactctac cagcagctga atgacttgga
agcctgtgtg 300 atgcaggagg agagggtggg agaaactccc ctgatgtacg
cggactccat cctggctgtg 360 aagaaatact tccaaagaat cactctctat
ctgacagaga agaaatacag cccttgtgcc 420 tgggaggttg tcagagcaga
aatcatgaga tccctctctt tatcaacaaa cttgcaagaa 480 agattaagga
ggaaggaata a 501 96 501 DNA human alpha interferon 96 tgtgatctgc
ctcagaccca cagcctgggt aataggaggg ccttgatact cctggcacaa 60
atgggaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg attcccccaa
120 gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct
ccatgagatg 180 atccagcaga ccttcaatct cttcagcaca aaggactcat
ctgctacttg ggaacagagc 240 ctcctagaaa aattttccac tgaacttaac
cagcagctga atgacatgga agcctgcgtg 300 atacaggagg ttggggtgga
agagactccc ctgatgaatg tggactctat cctggctgtg 360 aagaaatact
tccaaagaat cactctttat ctgacagaga agaaatacag cccttgtgct 420
tgggaggttg tcagagcaga aatcatgaga tccttctctt tatcaaaaat ttttcaagaa
480 agattaagga ggaaggaatg a 501 97 501 DNA human alpha interferon
97 tgtgatctgc ctcagaccca cagcctgggt aataggaggg ccttgatact
cctggcacaa 60 atgggaagaa tctctccttt ctcctgcctg aaggacagac
ctgactttgg acttccccag 120 gaggagtttg atggcaacca gttccagaag
actcaagcca tctctgtcct ccatgagatg 180 atccagcaga ccttcaatct
cttcagcaca gaggactcat ctgctgcttg ggaacagagc 240 ctcctagaaa
aattttccac tgaactttac cagcaactga ataacctgga agcatgtgtg 300
atacaggagg ttgggatgga agagactccc ctgatgaatg aggactccat cttggctgtg
360 aggaaatact tccaaagaat cactctttat ctaacagaga agaaatacag
cccttgtgcc 420 tgggaggttg tcagagcaga aatcatgaga tctctctctt
tttcaacaaa cttgcaaaaa 480 agattaagga ggaaggattg a 501 98 501 DNA
human alpha interferon 98 tgtgatctgc ctcagactca cagcctgggt
aataggaggg ccttgatact cctggcacaa 60 atgggaagaa tctctcattt
ctcctgcctg aaggacagat atgatttcgg attcccccag 120 gaggtgtttg
atggcaacca gttccagaag gctcaagcca tctctgcctt ccatgagatg 180
atccagcaga ccttcaatct cttcagcaca aaggattcat ctgctgcttg ggatgagacc
240 ctcctagaca aattctacat tgaacttttc cagcaactga atgacctaga
agcctgtgtg 300 acacaggagg ttggggtgga agagattgcc ctgatgaatg
aggactccat cctggctgtg 360 aggaaatact ttcaaagaat cactctttat
ctgatggaga agaaatacag cccttgtgcc 420 tgggaggttg tcagagcaga
aatcatgaga tccttctctt tttcaacaaa cttgcaaaaa 480 ggattaagaa
ggaaggattg a 501 99 11 PRT Artificial Sequence Description of
Artificial Sequence Protease peptide substrate 99 Arg Gly Val Val
Asn Ala Ser Ser Arg Leu Ala 1 5 10 100 44 DNA Artificial Sequence
Description of Artificial Sequence Introduced Sfi I site 100
ttccatttca tacatggccg aaggggccgt gccatgagga tttt 44 101 50 DNA
Artificial Sequence Description of Artificial Sequence Introduced
sfi I site 101 ttctaaatgc atgttggcct ccttggccgg attctgagcc
ttcaggacca 50
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